U.S. patent application number 10/116823 was filed with the patent office on 2003-02-06 for method for computer-assisted isolation and characterization of proteins.
Invention is credited to Anderson, N. Leigh, Anderson, Norman G., Goodman, Jack.
Application Number | 20030026465 10/116823 |
Document ID | / |
Family ID | 25379154 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030026465 |
Kind Code |
A1 |
Anderson, N. Leigh ; et
al. |
February 6, 2003 |
Method for computer-assisted isolation and characterization of
proteins
Abstract
The present invention provides an integrated, fully automated,
high-throughput system for two-dimensional electrophoresis
comprised of gel-making machines, gel processing machines, gel
compositions and geometries, gel handling systems, sample
preparation systems, software and methods. The system is capable of
continuous operation at high-throughput to allow construction of
large quantitative data sets.
Inventors: |
Anderson, N. Leigh;
(Washington, DC) ; Anderson, Norman G.;
(Rockville, MD) ; Goodman, Jack; (Lusby,
MD) |
Correspondence
Address: |
ROYLANCE, ABRAMS, BERDO & GOODMAN, L.L.P.
1300 19TH STREET, N.W.
SUITE 600
WASHINGTON,
DC
20036
US
|
Family ID: |
25379154 |
Appl. No.: |
10/116823 |
Filed: |
April 4, 2002 |
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Application
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10116823 |
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09642247 |
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6438259 |
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09339164 |
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6245206 |
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09339164 |
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09339165 |
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6136173 |
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6391650 |
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5993627 |
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Current U.S.
Class: |
382/129 |
Current CPC
Class: |
Y10T 436/113332
20150115; G01N 27/44773 20130101; G01N 2001/288 20130101; Y10T
436/25375 20150115; Y10T 436/25 20150115; G01N 27/44782 20130101;
G01N 27/44795 20130101; Y10T 436/255 20150115 |
Class at
Publication: |
382/129 |
International
Class: |
G06K 009/00 |
Claims
What is claimed is:
1. A method for preparing a sample protein solution for
electrophoresis analysis, said method comprises steps of: a)
preparing a column, which comprises of a first end, a second end
and a size exclusion material, by equilibrating said size exclusion
material with a first solvent; b) introducing a reagent into said
first end of said column to create a reagent zone; c) introducing a
protein sample into said first end of said column to create a
sample zone, wherein said sample contains a plurality of protein
molecules; d) allowing said protein molecules to flow through said
column, wherein said protein molecules flow ahead of said sample
zone, pass through said reagent zone, and pass into said first
solvent while in said column; and e) collecting said protein
molecules from said second end of said column.
2. The method according to claim 1, wherein said protein molecules
comprise a plurality of sulfhydryl groups which chemically react
with said reagent.
3. The method according to claim 2, wherein said sulfhydryl groups
covalently react with a zwitterionic or uncharged alkylating agent,
preventing oxidation of said sulfhydryl groups, and preserving an
isoelectric point of each of said protein molecules.
4. The method of claim 2, wherein said sulfhydryl groups are
reacted stoichiometrically with a negatively-charged alkylating
reagent, thereby shifting the isoelectric points of basic proteins
towards neutrality.
5. The method according to claim 1 wherein said protein molecules
react with one or more fluorescent or optically absorbing dyes in
said reagent zone, thereby said protein molecules are rendered
detectable by an optical means during and after subsequent
separation of said protein molecules.
6. The method according to claim 3, wherein said reagent is
S+2-amino-5-iodacetamido-pentanoic acid, iodoacetamide, iodoacetic
acid, N-ethyl maleimide or a combination thereof.
7. An apparatus for preparing a gel medium for separating a
plurality of protein molecules, said apparatus comprising: a) a
movable mold half which, when placed near a gel-binding material,
forms a mold cavity; b) means for moving said movable mold half; c)
means for transporting said gel-binding material; and d) means for
dispensing a polymerizable gel mixture into said mold cavity.
8. The apparatus of claim 7 wherein an O-ring is between said
movable mold half and said gel-binding material and wherein said
O-ring contacts both said movable mold half and said gel-binding
material.
9. The apparatus of claim 7 wherein said means for dispensing
comprises a means for dispensing a variable composition of said
polymerizable gel mixture, wherein a gradient of said gel mixture
is formed.
10. The apparatus of claim 7 wherein said gel mixture chemically
bonds to said gel-binding material.
11. The apparatus according to claim 7, wherein said apparatus
further comprises means for removing said gel-binding material from
said mold cavity.
12. The apparatus according to claim 7, wherein said means for
dispensing said polymerizable gel mixture comprises a precision
gradient maker, wherein said gradient maker comprises a plurality
of syringes coupled to a plurality of valves and said valves are
coupled to a plurality of reservoirs.
13. The apparatus according to claim 12, wherein said precision
gradient maker further comprises: a) a delivery tube comprising a
first end and a second end, wherein said first end is coupled to
said plurality of valves; and b) said second end of said delivery
tube is inserted into said mold cavity.
14. The apparatus according to claim 12, wherein said precision
gradient maker further comprises: a) a delivery tube comprising a
first end and a second end, wherein said first end is coupled to
said plurality of valves; and b) one of said valves coupled to said
second end so that said delivery tube can be alternatively coupled
to a lowest point in said mold cavity or coupled to a waste
container.
15. The apparatus according to claim 12, wherein each of said
reservoirs contains a solution selected from the group comprising:
a wash solution, one of a plurality of acrylamide monomer
solutions, an ammonium persulfate solution and a
tetramethylethylene diamine solution.
16. The apparatus according to claim 7 further comprising means for
controlling temperature of said gel mixture within said mold
cavity.
17. The apparatus according to claim 13 wherein said valves are
coupled to said reservoirs such that when said valves are switched
to allow refilling of said syringes said delivery tube is washed
with a non-polymerizable solvent.
18. The apparatus according to claim 17, wherein said apparatus
further comprises a means for aspirating a solution from said
delivery tube.
19. The apparatus according to claim 13 wherein further comprising
means for smoothly withdrawing said delivery tube during liquid
delivery so that said second end of said delivery tube remains at
or just above the rising meniscus of liquid in said mold
cavity.
20. A method of preparing a gel medium for separating a plurality
of protein molecules, wherein said method comprises steps of: a)
attaching a gel-binding material to a longitudinal mold cavity; b)
dispensing a variable composition of a polymerizable gel mixture
into said mold cavity through a delivery tube having an open end,
wherein said open end is inserted into said mold cavity, wherein
said gel mixture comprises a gradient and wherein said gel mixture
becomes chemically bonded to said backing material; c) withdrawing
said delivery tube from said mold cavity during said dispensing
whereby said open end of said delivery tube remains at or just
above the rising meniscus of said gel mixture in said mold cavity;
and d) removing said gel-binding material and said gel mixture from
said mold cavity.
21. The method according to claim 20, wherein said gel medium is
prepared and used for the analysis of a single sample, wherein said
sample comprises of said protein molecules.
22. The method according to claim 20, wherein said gel median has a
pH gradient along its length, rending said gel medium suitable for
use as an IPG gel.
23. The method according to claim 20, wherein said delivery tube is
washed with a non-polymerizable solvent composition thereby
preventing polymerization of said gel mixture in said delivery
tube.
24. The method according to claim 20, wherein said gradient is
dispensed as part of a sequence of segments comprising: a) a first
segment similar in volume to volume of said delivery tube, wherein
said first segment is dispensed before insertion of said delivery
tube into said mold cavity; b) a second segment comprising the
remainder of said gradient, wherein said segment is dispensed after
insertion of said delivery tube into said mold; and c) a third
segment having a volume similar to volume of said first
segment.
25. A method of preparing a gel medium for separating a plurality
of protein molecules, wherein said method comprises steps of: a)
attaching a backing material to a longitudinal mold cavity; b)
dispensing a variable composition of a polymerizable gel mixture
into said mold cavity, wherein said gel mixture comprising a
gradient, said gel mixture chemically bonding to said backing
material, and said mold cavity having a longitudinal portion with a
nonrectangular cross-section; and c) removing said backing material
and said gel mixture from said mold cavity.
26. The method according to claim 25, wherein said gel medium is
prepared and used for analyzing a single sample, wherein said
sample comprises said protein molecules.
27. A gel assembly for separating a plurality of proteins wherein
said gel assembly comprises: a) a gel comprising a longitudinal
gradient of titratable gel monomers; b) a strip of backing material
to which said gel is chemically bonded, said backing material
having a greater width than said gel; and c) a longitudinal groove
or cavity in which a liquid sample is held during use by capillary
forces.
28. A method of separating a plurality of proteins where said
proteins are in a liquid sample, said method comprising steps of:
a) applying said liquid sample onto a surface of a gel medium,
wherein said gel medium comprising a pH gradient and is attached to
a backing material; b) applying a voltage across said gel medium to
effect a separation of said proteins; c) subjecting said gel medium
to conditions wherein the volume of said liquid sample is
substantially decreased through imbibition of said liquid sample
into said gel medium or through loss of water in said liquid sample
to an insulating gaseous or a liquid environment of said gel, such
that said proteins are substantially incorporated into said
gel.
29. The method of claim 28 wherein said sample is applied to a
surface of said gel wherein said surface comprises a hole internal
to said gel, a groove formed in said surface of said gel, or a
groove comprising an included angle between said gel and an area of
said backing material extending outwardly from said gel.
30. An apparatus for processing a gel medium for separating a
plurality of protein molecules wherein said gel medium is fixed to
a backing material, wherein said apparatus comprises: a) a
plurality of stations; b) means for loading said gel medium with
said protein molecules; c) means for reversibly grasping said
backing material; and d) means for transporting said gel medium to
said stations.
31. The apparatus of claim 30, wherein one or more of said stations
comprise means for washing, dehydrating and rehydrating said gel
medium.
32. The apparatus of claim 30, wherein one or more of said stations
comprise means for application of a voltage longitudinally across
said gel medium.
33. The apparatus according to claim 30 further comprising means
for holding said gel medium in place at each of said stations.
34. The apparatus of claim 30, wherein each of said stations
comprises a plurality of slots into which a plurality of gel
mediums may be inserted.
35. The apparatus according to claim 32, wherein one or more of
said stations is maintained at different voltages.
36. A gel medium for separating a plurality of protein molecules,
wherein said gel medium comprises: a) a first segment, wherein said
first segment is planar and provides a medium for separating said
protein molecules; and b) a second segment, wherein said second
segment is of greater thickness than said first segment and
comprises a buffer reservoir for supplying ions.
37. The gel medium according to claim 36, wherein a rigid electrode
is embedded within said second segment and wherein said electrode
is used to apply a voltage across said gel.
38. The gel medium according to claim 37, wherein said electrode
forms a handle for transporting said gel medium.
39. The gel medium according to claim 36, wherein an internal slot
is formed in said second segment during molding, the floor of said
slot being at or near a junction between said first segment and
said second segment.
40. The gel medium according to claim 36 wherein a shallow external
slot is formed in said first segment during molding, said slot
running parallel to and nearby a junction between said first
segment and said second segment.
41. A gel medium according to claim 36 further comprising a third
segment, wherein said third segment is interposed between said
first segment and said second segment, said third segment having
characteristics of a stacking gel, wherein said protein molecules
are stacked between a first low molecular weight ionic species and
a second low molecular weight ionic species prior to effecting a
separation of said protein molecules in said first segment.
42. The gel medium of claim 36 further comprising a third segment
wherein said third segment is interposed between said first segment
and said second segment, and wherein said third segment comprises a
composition different from the compositions of said first segment
and said second segment and wherein said third segment has greater
strength and elasticity than said first segment and said second
segment.
43. A gel medium for separating a plurality of protein molecules,
wherein said gel medium comprises two regions of distinct geometry
wherein a first region is essentially planar and provides a medium
for separating a plurality of macromolecules and a second region is
non-planar and provides a means for suspending said gel by an edge
in liquid or gaseous surroundings.
44. The gel medium according to claim 43, wherein part of said gel
medium is polymerized around a rigid support.
45. The gel medium according to claim 44, wherein said rigid
support is used to transport or suspend said gel medium.
46. The gel medium according to claim 43, wherein said second
region is formed with projections or indentations which facilitate
grasping of said second region when transporting or holding said
gel medium.
47. The gel medium according to claim 43 further comprising a third
region wherein said third region is interposed between said first
region and second region, and wherein said third region has a
different composition than said first region and said second region
and said third region displays greater strength and elasticity than
said first region and said second region.
48. The gel medium according to claim 43, wherein a rigid electrode
is polymerized within said second region, said electrode extends
outside of said second region at one or more locations and said
electrode is used to apply a voltage across said gel medium.
49. A method of performing electrophoreses in an acrylamide gel,
said method comprises steps of: a) polymerizing said gel in a mold;
b) removing said gel from said mold by reversible mechanical
interaction with a region of said gel; and c) performing an
electrophoretic separation in said gel.
50. The method according to claim 49 wherein said gel is supported
by means of a rigid mechanical component, wherein said component is
at least partially enclosed within said gel.
51. The method according to claim 49 wherein said gel comprises a
first segment in which macromolecular separation occurs and a
second segment comprising a first buffer reservoir.
52. The method according to claim 51 wherein a sample is applied to
said gel by inserting said sample into a slot inside said gel such
that the floor of said slot is at or near a junction between said
first segment and said second segment.
53. The method according to claim 51 wherein a sample is applied to
an external surface of said gel nearby a junction between said
first segment and said second segment.
54. The method according to claim 51 wherein said second segment
partially encloses a rigid electrode.
55. The method according to claim 54 wherein said first segment
comprises a first end which contacts said second segment and a
second end which is distal from said second segment and wherein
said second end contacts a second buffer reservoir at an electrical
voltage different from a voltage applied to said rigid
electrode.
56. The method according to claim 55 wherein said gel is at least
partially suspended in an insulating fluid.
57. The method according to claim 56 wherein said insulating fluid
is less dense than fluid comprising said second buffer
reservoir.
58. The method according to claim 57 wherein said insulating fluid
is cooled.
59. The method according to claim 58 wherein said insulating fluid
is circulated over a surface of said gel.
60. The method according to claim 51 wherein said gel further
comprises a third segment which is a stacking gel.
61. The method according to claim 49 wherein said gel comprises a
plurality of projections or cavities or a combination thereof for
mechanically supporting said gel.
62. The method according to claim 61 wherein said gel comprises a
first segment in which macromolecular separation occurs and a
second segment comprising a first buffer reservoir, and wherein
said first segment comprises a first end which contacts said second
segment and a second end which is distal from said second segment
and wherein said second end contacts a second buffer reservoir at
an electrical voltage different from a voltage applied to said
rigid electrode.
63. The method according to claim 62 wherein said gel is at least
partially suspended in an insulating fluid during
electrophoresis.
64. The method according to claim 63 wherein said insulating fluid
is less dense than fluid comprising said second reservoir
buffer.
65. The method according to claim 64 wherein said insulating fluid
is cooled.
66. The method according to claim 65 wherein said insulating fluid
is circulated over the surface of said gel.
67. The method according to claim 49 wherein said gel is grasped by
a plurality of gripping movable jaws, wherein one or more of said
jaws comprises a cavity and an electrode, said cavity forming a
liquid vessel bounded in part by said gel, such that when said
vessel is filled with an appropriate buffer solution, electrical
contact is established between said electrode and said gel.
68. The method according to claim 67, wherein an edge of said gel
is distal from said liquid vessel, said edge contacting a second
buffer reservoir at an electrical voltage different from a voltage
applied to said electrode.
69. The method according to claim 68 wherein said get is at least
partially suspended in an insulating fluid.
70. The method according to claim 67 wherein each of said gripping
movable jaws has a first face and a second face, each of said first
face and said second face being in frictional contact with said
gel.
71. The method according to claim 70, wherein each of said first
face and said second face comprises a plurality of small sharp grit
particles.
72. The method according to claim 49, wherein said gel is grasped
by a plurality of gripping movable jaws, wherein one or more of
said jaws comprises an internal liquid channel having an external
input and an external output, said channel having at least one
segment exposed to the surface of said gel such that a liquid
circulating through said channel contacts said gel.
73. The method according to claim 72, wherein an edge of said gel
is distal from said liquid channel, said edge contacting a second
buffer reservoir at an electrical voltage different from a voltage
applied to said electrode.
74. The method according to claim 73 wherein said gel is at least
partially suspended in an insulating fluid.
75. The method according to claim 72, wherein each of said gripping
movable jaw has a first face and a second face, each of said first
face and said second face being in frictional contact with said
gel.
76. The method according to claim 75, wherein each of said first
face and said second face comprises a plurality of small sharp grit
particles.
77. The method of detecting macromolecules in an electrophoreses
gel which method comprises the step of suspending said gel by an
edge of said gel in a solution or a sequence of solutions such that
said macromolecules are rendered detectable.
78. The method according to claim 77, wherein said gel is
transferred from one solution to another by a movable arm.
79. The method according to claim 77, wherein said gel is suspended
by a rigid member which is at least partially embedded within said
gel.
80. The method according to claim 77, wherein said gel is suspended
from a non-planar region of said gel.
81. The method according to claim 77, wherein said gel is suspended
by a plurality of gripping movable jaws.
82. The method according to claim 81, wherein said jaws comprise
springs, magnets, electrical solenoids, pneumatic pistons or
hydraulic pistons.
83. The method according to claim 81, wherein each of said jaws has
a first face and a second face, each of said first face and said
second face being in frictional contact with said gel.
84. The method according to claim 83, each of said first face and
second face comprises a plurality of small sharp grit
particles.
85. A method of detecting macromolecules in an electrophoresis gel
which method comprises the step of placing said gel in a holder
having an internal cavity with dimensions similar to said gel, and
suspending said gel in a solution or a sequence of solutions such
that said macromolecules are rendered detectable.
86. A method for scanning a stained gel medium, wherein said method
comprises the steps of: a) grasping an edge of said gel medium; b)
transporting said gel medium to a scanning station by a mechanical
motion means; and c) placing said gel medium in a space illuminated
by a light source and within view of a light detector.
87. The method according to claim 86 further comprising the step of
detecting light absorption, light scatter, fluorescence,
luminescence, or fluorographic emission of said gel medium.
88. The method according to claim 86, wherein said gel medium is
scanned by a position-sensitive optical sensor, photodiode array
camera, CCD camera, moving laser beam, or a moving scanning
head.
89. The method according to claim 86, wherein said mechanical
motion means supports said gel by a rigid support embedded in said
gel.
90. The method according to claim 86, wherein said mechanical
motion means supports said gel by interaction with a non-planar
region of said gel.
91. The method according to claim 86, wherein said mechanical
motion means grasps said edge of said gel by a plurality of
gripping movable jaws.
92. A method of scanning a stained electrophoresis gel, said method
comprises the steps of: a) immersing said gel in a thin planar
cavity filled with a liquid having a refractive index similar to
that of said gel; b) introducing an illuminating light into said
cavity approximately in the plane of said gel, wherein said
illuminating light is substantially internally reflected in said
cavity and thereby prevented from exiting said cavity normal to a
plane of said gel; and c) positioning an optical scanner such that
said gel is viewed from outside of said cavity along a line of
sight normal to said gel.
93. The method according to claim 92, wherein said cavity is a
shallow horizontal depression filled with said liquid, wherein said
liquid is aqueous.
94. The method according to claim 92 further comprising the step of
positioning a light absorbing surface on an opposite side of said
gel from said optical scanner, wherein said gel is stained with a
fluorescent dye.
95. The method according to claim 94, wherein said illuminating
light comprises a spectrum enriched in an appropriate excitation
wavelength of said dye and depleted of an appropriate emission
wavelength of said dye, and said light impinges on said optical
scanner after passage through a filter which preferentially absorbs
said excitation wavelength and transmits said appropriate emission
wavelength of said dye.
96. The method according to claim 92 further comprising the steps
of: a) positioning a light absorbing surface on an opposite side of
said gel from said scanner; and b) differentiating a plurality of
stained features on said gel from background by a greater or a
lesser scattering of said illuminating light in a direction of said
scanner, wherein said gel is stained with a particulate stain.
97. A method of establishing relative quantitation of proteins
resolved in an electrophoresis gel, said method comprising steps
of: a) staining said gel such that a plurality of optical
properties of a plurality of protein-containing regions in said gel
are progressively changed during a period of time; b) optically
scanning said gel two or more times during said period; c)
measuring each of said optical properties as a function of time and
recording a time sequence for said optical properties; d) deriving
a mathematical index from said time sequence; and e) calculating
relative protein abundance of said gel or other useful property of
said gel from said index.
98. The method according to claim 97, wherein said gel is stained
using a silver-based stain, a negative stain based on interaction
of a detergent or a plurality of ions in said gel with a plurality
of copper ions or a plurality of zinc ions in said gel, a Schiff
stain for carbohydrates, or other kinetic stain, wherein a
plurality of rates of diffusion of a plurality of reactants or a
plurality of rates of chemical reactions determine said period of
time.
99. The method according to claim 98, wherein said optical
properties comprise transmittance, absorbance, fluorescence,
luminescence, light scatter or refractive index as a function of
time.
100. The method according to claim 98, wherein said mathematical
index comprises a maximum rate of change of said optical properties
as a function of time, or an increment of time relative to a fixed
time at which a given change in said optical properties is
detected, or a combination of said rate of change and said
increment with one or more of said optical properties.
101. A method establishing relative quantitation of proteins
resolved in an electrophoresis gel wherein: a) staining said gel by
two or more staining procedures to reveal said proteins; b)
optically scanning said gel to detect a plurality of stain results
after or during each of said staining procedure; and c)
collectively interpreting said stain results by means of a
plurality of mathematical equations to determine a single measure
of protein abundance.
102. The method of comparing the properties of a plurality of
proteins which may be present in a plurality of samples, wherein:
a) each of said samples is reacted with a protein labeling reagent
capable of being detected separately from labeling reagents used to
label other samples to be compared; b) said samples are combined
and said proteins they contain are separated by one or more
separations processes; c) each of said labeling reagents is
quantitatively measured at a plurality of points along said
separation to yield a protein profile for each sample; and d) said
profiles are compared by a mathematical technique to estimate the
likelihood that apparently co-separating proteins in different
samples are identical.
103. The method of claim 102 wherein each sample to be combined is
labeled with a fluorescent dye having a different emission
spectrum.
104. The method of claim 102 wherein said mathematical technique
compares said profiles by correlation or by means sensitive to peak
width, shape or position.
105. The method of claim 102 wherein said likelihoods of protein
identity provide a means of detecting non-identity that is more
sensitive than the resolving power of said separations process to
resolve two proteins in a single sample.
106. A method of excising a plurality of small regions of an
electrophoresis gel comprising resolved proteins, said method
comprising steps of: a) staining said gel; b) scanning said gel to
yield a digitized image; c) inputting said image into an image
processor for identifying a plurality of protein-containing regions
in said gel; d) excising said protein-containing regions from said
gel by means of a computer-controlled movable punch device on the
basis of coordinates supplied by said image processor, wherein said
punch device comprises a tube-like cutter and a central piston
within said cutter, said cutter and said piston being moved coaxial
by a computer-controlled means to cut, retrieve and expel a plug of
said gel; and e) depositing said protein-containing regions in a
plurality of vessels.
107. A system for preparing an electrophoresis gel, wherein said
system comprises a computer and a software, and said computer and
software control a plurality of electromechanical means for
preparing said gel and for removing said gel from a mold.
108. The system according to claim 107, wherein said system further
loads a sample onto said gel, wherein said sample comprises a
plurality of components.
109. The system according to claim 108, wherein said system further
effects an electrophoretic separation of said components within
said electrophoresis gel.
110. The system according to claim 109, wherein said system further
detects said components after electrophoresis and staining of said
gel.
111. The system according to claim 110, wherein said system
performs a two dimensional protocol by preparing a first gel and a
second gel, loading said sample onto said first gel, effecting a
first separation of said components in said first gel, transferring
said first gel to said second gel, effecting a second separation of
said components in said second gel, and detecting said
components.
112. A method for preparing an electrophoresis gel, said method
comprises steps of: casting said gel in a mold; and removing said
gel from said mold by a plurality of electromechanical means,
wherein each of said electromechanical means is controlled by a
computer and a software.
113. The method according to claim 112, wherein said computer and
said software further control loading of a sample onto said gel,
wherein said sample comprises a plurality of components.
114. The method according to claim 113, wherein said computer and
said software further control detection of said components within
said gel.
115. The method according to claim 114, wherein said computer and
said software further control detection of said components within
said gel.
116. The method according to claim 115, wherein said computer and
said software control: 1) preparation of a first gel and a second
gel, 2) loading of said sample onto said first gel, 3) a first
separation of said components in said first gel, 4) transferring of
said first gel to said second gel, 5) a second separation of said
components in said second gel, and 6) detection of said components
within said gel.
117. A method for separating a plurality of protein molecules in a
plurality of gel mediums, said method comprising steps of: a)
grasping an edge of each of said gel mediums; b) transporting each
of said gel mediums to a plurality of stations wherein said
stations comprise a plurality of environments; and c) inserting
each of said gel mediums into said stations for separating said
protein molecules.
118. The method according to claim 117, wherein said gel mediums
are held by a plurality of grasping means respectively.
119. The method according to claim 118, wherein said grasping means
comprise one or more springs, magnets, electrical solenoids,
pneumatic pistons, or hydraulic pistons.
120. A programmable system for separating a plurality of protein
molecules in a plurality of gel mediums respectively, said system
comprising: a) means for preparing said gel mediums; b) means for
leading each of said gel mediums with said protein molecules; c)
means for separating said protein molecules in each of said gel
mediums; d) means for staining or otherwise revealing proteins in
each of said gels; and e) means for scanning each of said gel
mediums to detect resolved macromolecules.
121. The programmable system according to claim 120 further
comprising a plurality of stations.
122. The programmable system according to claim 121, wherein each
of said gel mediums is assigned thereto a plurality of time
intervals for each of said stations respectively.
123. The programmable system according to claim 121, wherein each
of said gel mediums is assigned thereto a plurality of parameters
for separating said protein molecules in each of said gel
mediums.
124. The programmable system according to claim 122 further
comprising means for scheduling said time intervals for each of
said stations respectively.
125. The programmable system according to claim 124, wherein said
means for scheduling is asynchronous.
126. The programmable system according to claim 122 wherein said
time intervals assigned to at least one of said gel mediums is
different from said time intervals assigned to at least a second of
said gel mediums.
127. The programmable system according to claim 123 further
comprising means for controlling said plurality of parameters
assigned to each of said gel mediums.
128. The programmable system according to claim 123 wherein said
parameters comprise volt-hours.
129. The programmable system according to claim 123 wherein said
parameters comprise a pH gradient of said gel mediums.
130. The programmable system according to claim 123, wherein said
parameters assigned to at least one of said gel mediums are
different from said parameters assigned to at least a second of
said gel mediums.
131. The programmable system according to claim 123 further
comprising means for grasping each of said gel mediums at each of
said stations.
132. The programmable system according to claim 123 further
comprising means for transporting each of said gel mediums to said
stations.
133. The programmable system according to claim 122 further
comprising a database, wherein a first plurality of steps and a
first plurality of execution times based on said time intervals for
a first sample are entered into said database, and a second
plurality of steps and a second plurality of execution times based
on said time intervals for a second sample are entered into said
database, said second plurality of steps including a start time
delay calculated so as to prevent any action required for said
second sample from interfering with any action required for said
first sample, and said system retrieves said first and second
plurality of steps and said first and second plurality of execution
times from said databases and carries out first and second
plurality of steps in time order.
134. An integrated system for two-dimensional electrophoresis, said
system comprising: a) means for preparing an isoelectric focusing
gel; b) means for loading said isoelectric focusing gel with a
plurality of protein molecules; c) means for applying a first
voltage across said isoelectric focusing gel; d) means for
preparing a slab electrophoresis gel; e) means for loading said
isoelectric focusing gel onto said slab electrophoresis gel; f)
means for applying a second voltage across said slab
electrophoresis gel; and g) means for staining said slab
electrophoresis gel.
135. The integrated system according to claim 134, wherein said
system is programmable.
136. The integrated system according to claim 135, wherein said
system can operate on a repeated basis.
137. The integrated system according to claim 135, wherein said
system comprises means for scanning said gel.
138. The integrated system according to claim 137, wherein said
system comprises computer software means for extracting estimates
of protein abundance and position from an image generated by said
means for scanning said gel.
139. The integrated system according to claim 137, wherein said
system comprises computer software means for inserting said
estimates of protein abundance and position into a database.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 09/642,247, which is a Divisional of U.S.
patent application Ser. No. 09/642,246, both filed Aug. 17, 2000,
and both of which are Divisionals of U.S. patent application Ser.
No. 09/339,164, which is a Divisional of U.S. patent application
Ser. No. 09/339,165, now U.S. Pat. No. 6,136,173, issued Oct. 24,
2000, which is a Divisional of U.S. patent application Ser. No.
09/339,177, all of which were filed Jun. 24, 1999, and all of which
are Divisionals of U.S. patent application Ser. No. 08/881,761,
filed Jun. 24, 1997, now U.S. Pat. No. 5,993,627, issued Nov. 30,
1999.
BACKGROUND OF THE INVENTION
[0002] The invention relates to the field of electrophoretic
separations of macromolecules and in particular, to the automation
of two-dimensional electrophoretic separations used in the analysis
of proteins. Such two-dimensional procedures typically involve
sequential separations by isoelectric focusing (IEF) and SDS slab
gel electrophoresis, and an automated 2-D method thus involves
manufacture and use of gel media for both isoelectric focusing and
SDS electrophoresis, together with means for protein detection and
quantitation. Two-dimensional electrophoresis technology forms the
basis of the expanding field of proteomics, and hence automation of
the procedure is a critical requirement for scale-up of efforts to
build proteome databases comprising all the proteins of complex
organisms such as man. To date, no successful automation efforts
have been reported, despite the use of bench-scale 2-D
electrophoresis in more than 5,000 scientific publications.
[0003] The publications and other materials used herein to
illuminate the background of the invention and in particular, cases
to provide additional details respecting the practice, are
incorporated herein by reference, and for convenience are
referenced in the following text and respectively grouped in the
appended List of References. Elements of the invention are
disclosed in our Disclosure Documents 393753, 393754 and
412899.
[0004] Isoelectric Focusing (IEF)
[0005] A protein is a macromolecule composed of a chain of amino
acids. Of the 20 amino acids found in typical proteins, four
(aspartic and glutamic acids, cysteine and tyrosine) carry a
negative charge and three (lysine, arginine and histidine) a
positive charge, in some pH range. A specific protein, defined by
its specific sequence of amino acids, is thus likely to incorporate
a number of charged groups along its length. The magnitude of the
charge contributed by each amino acid is governed by the prevailing
pH of the surrounding solution, and can vary from a minimum of 0 to
a maximum of 1 charge (positive or negative depending on the amino
acid), according to a titration curve relating charge and pH
according to the pK of the amino acid in question. Under denaturing
conditions in which all of the amino acids are exposed, the total
charge of the protein molecule is given approximately by the sum of
the charges of its component amino acids, all at the prevailing
solution pH.
[0006] Two proteins having different ratios of charged, or
titrating, amino acids can be separated by virtue of their
different net charges at some pH. Under the influence of an applied
electric field, a more highly charged protein will move faster than
a less highly charged protein of similar size and shape. If the
proteins are made to move from a sample zone through a
non-convecting medium (typically a gel such as polyacrylamide), an
electrophoretic separation will result.
[0007] If, in the course of migrating under an applied electric
field, a protein enters a region whose pH has that value at which
the protein's net charge is zero (the isoelectric pH), it will
cease to migrate relative to the medium. Further, if the migration
occurs through a monotonic pH gradient, the protein will "focus" at
this isoelectric pH value. If it moves toward more acidic pH
values, the protein will become more positively charged, and a
properly-oriented electric field will propel the protein back
towards the isoelectric point. Likewise, if the protein moves
towards more basic pH values, it will become more negatively
charged, and the same field will push it back toward the
isoelectric point. This separation process, called isoelectric
focusing, can resolve two proteins differing by less than a single
charged amino acid among hundreds in the respective sequences.
[0008] A key requirement for an isoelectric focusing procedure is
the formation of an appropriate spatial pH gradient. This can be
achieved either dynamically, by including a heterogeneous mixture
of charged molecules (ampholytes) into an initially homogeneous
separation medium, or statically, by incorporating a spatial
gradient of titrating groups into the gel matrix through which the
migration will occur. The former represents classical
ampholyte-based isoelectric focusing, and the latter the more
recently developed immobilized pH gradient (IPG) isoelectric
focusing technique. The IPG approach has the advantage that the pH
gradient is fixed in the gel, while the ampholyte-based approach is
susceptible to positional drift as the ampholyte molecules move in
the applied electric field. The best current methodology combines
the two approaches to provide a system where the pH gradient is
spatially fixed but small amounts of ampholytes are present to
decrease the adsorption of proteins onto the charged gel matrix of
the IPG.
[0009] It is current practice to create IPG gels in a thin planar
configuration bonded to an inert substrate, typically a sheet of
Mylar plastic which has been treated so as to chemically bond to an
acrylamide gel (e.g., Gelbond.RTM. PAG film, FMC Corporation). The
IPG gel is typically formed as a rectangular plate 0.5 mm thick, 10
to 30 cm long (in the direction of separation) and about 10 cm
wide. Multiple samples can be applied to such a gel in parallel
lanes, with the attendant problem of diffusion of proteins between
lanes producing cross contamination. In the case where it is
important that all applied protein in a given lane is recovered in
that lane (as is typically the case in 2-D electrophoresis), it has
proven necessary to split the gel into narrow strips (typically 3
mm wide), each of which can then be run as a separate gel. Since
the protein of a sample is then confined to the volume of the gel
represented by the single strip, it will all be recovered in that
strip. Such strips (Immobiline DryStrips) are produced commercially
by Pharmacia Biotech.
[0010] While the narrow strip format solves the problem of
containing samples within a recoverable, non-cross-contaminating
region, there remain substantial problems associated with the
introduction of sample proteins into the gel. Since
protein-containing samples are typically prepared in a liquid form,
the proteins they contain must migrate, under the influence of the
electric field, from a liquid-holding region into the IPG gel in
order to undergo separation. This is typically achieved by lightly
pressing an open-bottomed rectangular frame against the planar gel
surface so that the gel forms the bottom of an open-topped but
otherwise liquid-tight vessel (the sample well). The sample is then
deposited in this well in contact with the gel surface forming the
bottom of the well. Since all of the sample protein must pass
through a small area on the surface of the gel (the well bottom) in
order to reach the gel interior, the local concentration of protein
at the entry point can become very high, leading to protein
precipitation. The sample entry area is typically smaller than the
gel surface forming the well bottom because the protein migrates
into the gel under the influence of an electric field which directs
most of it to one edge of the well bottom, tending to produce
protein precipitation. The major source of precipitation, however,
is provided by the charged groups introduced into the gel matrix to
form the pH gradient in IPG gels: these groups can interact with
charges on the proteins (most of which are not at their isoelectric
points at the position of the application point and hence have
non-zero net charges) to bind precipitates to the gel. It is common
experience that separations of the same protein mixture on a series
of apparently identical IPG gels can yield very different
quantitative recoveries of different proteins at their respective
isoelectric points, indicating that the precipitation phenomenon
may vary from gel to gel in unpredictable ways, thereby frustrating
the general use of IPG gels for quantitative protein
separations.
[0011] Recently, methods have been introduced in which the IPG
strip is re-swollen, from the dry state, in a solution containing
sample proteins, with the intention that the sample proteins
completely permeate the gel at the start of the run.
[0012] Isoelectric focusing separation of proteins in an
immobilized pH gradient (IPG) is extensively described in the art.
The concept of the IPG is disclosed in U.S. Pat. No. 4,130,470 and
is further described in numerous publications. The IPG gel strips
manufactured are generally of simple planar shape.
[0013] A series of disclosures have dealt with various
configurations of cavities ("sample wells") used for the
application of macromolecular-containing samples to the surfaces of
gels, most frequently slab gels used for protein or nucleic acid
separations. In each case, these sample wells were designed to
concentrate macromolecules in the sample into a thin starting zone
prior to their migration through the resolving gel. The following
references describe the use of devices placed against a gel to form
wells: U.S. Pat. No. 5,304,292 describes the use of pieces of
compressible gasket to form well walls at the top of a slab where
the ends of the pieces touch the top edge of the slab. U.S. Pat.
No. 5,164,065 describes a shark's tooth comb used in combination
with DNA sequencing gels.
[0014] Several references describe automated devices for creating
gradients of polymerizable monomers. Such systems have been used
for making porosity gradient gels used in molecular weight
separations of proteins. Altland, et al. (Altland, K. and Altland,
A. Pouring reproducible gradients in gels under computer control,
Clin. Chem. 30(12 Pt 1): 2098-2103, 1984) shows the use of such
systems for creating the gradients of titratable monomers used in
the creation of IPG gels. U.S. Pat. No. 4,169,036 describes a
system for loading slab-gel holders for electrophoresis separation.
U.S. Pat. No. 4,594,064 discloses an automated apparatus for
producing gradient gels. Hence, use of a computer-controlled
gradient maker in manufacturing IPG and other gels is known in the
art.
[0015] One alternative method of running IPG strips in an IsomorpH
device is disclosed in Disclosure Document No. 342751 (Anderson, N.
L., entitled "Vertical Method for Running IPG Gel Strips"). The
disclosed device uses sample wells pressed against the gel surface,
but otherwise completely closed, so that the assembly could be
rotated into a vertical orientation, thus allowing closer packing
of gels and a greater gel capacity in a small instrument footprint.
Additional methods are disclosed in Disclosure Document No's 393753
(Anderson, N. L., Goodman, Jack, and Anderson, N. G., entitled "Gel
Strips for Protein Separation") and 412899 (Anderson, N. L.,
Goodman, Jack, and Anderson, N. G., entitled "Automated System for
Two-Dimensional Electrophoresis").
[0016] Systems for making non-planar slab gels are also known in
the art and are disclosed in the following references: U.S. Pat.
No. 5,074,981 discloses a substitute for submarine gels using an
agarose block that is thicker at the ends and hangs into buffer
reservoirs. U.S. Pat. No. 5,275,710 discloses lane-shaped gels
formed in a plate and gel-filled holes extending down from the
plate into buffer reservoirs. These gel systems, however, do not
provide a gel which can be given a cross-section that is optimal
for producing high-resolution protein separation. Furthermore,
these systems cannot incorporate varying cross-sections along the
length of a gel as required.
[0017] SDS Slab Gel Electrophoresis
[0018] Charged detergents such as sodium dodecyl sulfate (SDS) can
bind strongly to protein molecules and "unfold" them into
semi-rigid rods whose lengths are proportional to the length of the
polypeptide chain, and hence approximately proportional to
molecular weight. A protein complexed with such a detergent is
itself highly charged (because of the charges of the bound
detergent molecules), and this charge causes the protein-detergent
complex to move in an applied electric field. Furthermore, the
total charge also is approximately proportional to molecular weight
(since the detergent's charge vastly exceeds the protein's own
intrinsic charge), and hence the charge per unit length of a
protein-SDS complex is essentially independent of molecular weight.
This feature gives protein-SDS complexes essentially equal
electrophoretic mobility in a non-restrictive medium. If the
migration occurs in a sieving medium, such as a polyacrylamide gel,
however, large (long) molecules will be retarded compared to small
(short) molecules, and a separation based approximately on
molecular weight will be achieved. This is the principle of SDS
electrophoresis as applied commonly to the analytical separation of
proteins.
[0019] An important application of SDS electrophoresis involves the
use of a slab-shaped electrophoresis gel as the second dimension of
a two-dimensional procedure. The gel strip or cylinder in which the
protein sample has been resolved by isoelectric focusing is placed
along the slab gel edge and the molecules it contains are separated
in the slab, perpendicular to the prior separation, to yield a
two-dimensional (2-D) separation. Fortunately, the two parameters
on which this 2-D separation is based, namely isoelectric point and
mass, are almost completely unrelated. This means that the
theoretical resolution of the 2-D system is the product of the
resolutions of each of the constituent methods, which is in the
range of 150 molecular species for both IEF and SDS
electrophoresis. This gives a theoretical resolution for the
complete system of 22,500 proteins, which accounts for the intense
interest in this method. In practice, as many as 5,000 proteins
have been resolved experimentally. The present invention is aimed
primarily at the 2-D application, and includes means for automating
the second dimension SDS separation of a 2-D process to afford
higher throughput, resolution and speed.
[0020] It is current practice to mold electrophoresis slab gels
between two flat glass plates, and then to load the sample and run
the slab gel still between the same glass plates. The gel is molded
by introducing a dissolved mixture of polymerizable monomers,
catalyst and initiator into the cavity defined by the plates and
spacers or gaskets sealing three sides. Polymerization of the
monomers then produces the desired gel media. This process is
typically carried out in a laboratory setting, in which a single
individual prepares, loads and runs the gel. A gasket or form
comprising the bottom of the molding cavity is removed after gel
polymerization in order to allow current to pass through two
opposite edges of the gel slab: one of these edges represents the
open (top) surface of the gel cavity, and the other is formed
against its removable bottom. Typically, the gel is removed from
the cassette defined by the glass plates after the electrophoresis
separation has taken place, for the purposes of staining,
autoradiography, etc., required for detection of resolved
macromolecules such as proteins.
[0021] The concentrations of polyacrylamide gels used in
electrophoresis are stated generally in terms of % T (the total
percentage of acrylamide in the gel by weight) and % C (the
proportion of the total acrylamide that is accounted for by the
crosslinker used). N,N'-methylenebisacrylami- de ("bis") is
typically used as crosslinker. Typical gels used to resolve
proteins range from 8% T to 24% T, a single gel often incorporating
a gradient in order to resolve a broad range of protein molecular
masses.
[0022] In most conventional systems used for SDS electrophoresis,
use is made of the stacking phenomenon first employed in this
context by Laemmli, U.K. (1970) Nature 227, 680. In a stacking
system, an additional gel phase of high porosity is interposed
between the separating gel and the sample. The two gels initially
contain a different mobile ion from the ion source (typically a
liquid buffer reservoir) above them: the gels contain chloride (a
high mobility ion) and the buffer reservoir contains glycine (a
lower mobility ion, whose mobility is pH dependent). All phases
contain Tris as the low-mobility, pH determining other buffer
component and positive counter-ion. Negatively charged protein-SDS
complexes present in the sample are electrophoresed first through
the stacking gel at its pH of approximately 6.8, where the
complexes have the same mobility as the boundary between the
leading (Cl-) and trailing (glycine-) ions. The proteins are thus
stacked into a very thin zone "sandwiched" between Cl- and
glycine-zones. As this stacking boundary reaches the top of the
separating gel the proteins become unstacked because, at the higher
separating gel pH (8.6), the protein-SDS complexes have a lower
mobility. Thus, in the separating gel, the proteins fall behind the
stacking front and are separated from one another according to size
as they migrate through the sieving environment of the lower
porosity (higher % T acrylamide) separating gel. In this
environment, proteins are resolved on the basis of mass.
[0023] Pre-made slab gels have been available commercially for many
years (e.g., from Integrated Separation Systems). These gels are
prepared in glass cassettes much as would be made in the user's
laboratory, and shipped from a factory to the user. More recently,
methods have been devised for manufacture of both slab gels in
plastic cassettes (thereby decreasing the weight and fragility of
the cassettes) and slab gels bonded to a plastic backing (e.g.,
bonded to a Gelbond.RTM. Mylar.RTM. sheet or to a suitably
derivatized glass plate). To date, all commercially-prepared gels
are either enclosed in a cassette or bonded to a plastic sheet on
one surface (the other being covered by a removable plastic
membrane). Furthermore, all commercially-prepared gels have a
planar geometry.
[0024] Current practice in running slab gels involves one of two
methods. A gel in a cassette is typically mounted on a suitable
electrophoresis apparatus, so that one edge of the gel contacts a
first buffer reservoir containing an electrode (typically a
platinum wire) and the opposite gel edge contacts a second
reservoir with a second electrode, steps being taken so that the
current passing between the electrodes is confined to run mainly or
exclusively through the gel. Such apparatus may be "vertical" in
that the gel's upper edge is in contact with an upper buffer
reservoir and the lower edge is in contact with a lower reservoir,
or the gel may be rotated 90.degree. about an axis perpendicular to
its plane, so that the gel runs horizontally between a left and
right buffer reservoir, as is disclosed in U.S. Pat. No. 4,088,561
(e.g., "DALT" electrophoresis tank). Various configurations have
been devised in order to make these connections electrically, and
to simultaneously prevent liquid leakage from one reservoir to the
other (around the gel).
[0025] When used as part of a typical 2-D procedure, an EF gel is
applied along one exposed edge of such a slab gel and the proteins
it contains migrate into the gel under the influence of an applied
electric field. The IEF gel may be equilibrated with solutions
containing SDS, buffer and thiol reducing agents prior to placement
on the SDS gel, in order to ensure that the proteins the IEF gel
contains are prepared to begin migrating under optimal conditions,
or else this equilibration may be performed in situ by surrounding
the gel with a solution or gel containing these components after it
has been placed in position along the slab's edge.
[0026] A slab gel affixed to a Gelbond.RTM. sheet is typically run
in a horizontal position, lying flat on a horizontal cooling plate
with the Gelbond.RTM. sheet down and the unbonded surface up.
Electrode wicks communicating with liquid buffer reservoirs, or
bars of buffer-containing gel, are placed on opposite edges of the
slab to make electrical connections for the run, and samples are
generally applied onto the top surface of the slab (as in the
instructions for the Pharmacia ExcelGels).
[0027] It is current practice to detect proteins in 2-D gels either
by staining the gels or by exposing the gels to a radiosensitive
film or plate (in the case of radioactively labeled proteins).
Staining methods include dye-binding (e.g., Coomassie Brilliant
Blue), silver stains (in which silver grains are formed in
protein-containing zones), negative stains in which, for example,
SDS is precipitated by Zn ions in regions where protein is absent,
or the proteins may be fluorescently labeled. In each case, images
of separated protein spot patterns can be acquired by scanners, and
this data reduced to provide positional and quantitative
information on sample protein composition through the action of
suitable computer software.
[0028] Additional methods are disclosed in Disclosure Document
No's. 393754 (Anderson, N. L., Goodman, Jack, and Anderson, N. G.,
entitled "Apparatus and Methods for Casting and Running
Electrophoresis Slab Gels") and 412899 (Anderson, N. L., Goodman,
Jack, and Anderson, N. G., entitled "Automated System for
Two-Dimensional Electrophoresis").
[0029] Sample Preparation
[0030] Protein samples to be analyzed using 2-D electrophoresis are
typically solubilized in an aqueous, denaturing solution such as 9M
urea, 2% NP-40 (a non-ionic detergent), 2% of a pH 8-10.5 ampholyte
mixture and 1% dithiothreitol (DTT). The urea and NP-40 serve to
dissociate complexes of proteins with other proteins and with DNA,
RNA, etc. The ampholyte mixture serves to establish a high pH
(.about.9) outside the range where most proteolytic enzymes are
active, thus preventing modification of the sample proteins by such
enzymes in the sample, and also complexes with DNA present in the
nuclei of sample cells, allowing DNA-binding proteins to be
released while preventing the DNA from swelling into a viscous gel
that interferes with IEF separation. The purpose of the DTT is to
reduce disulfide bonds present in the sample proteins, thus
allowing them to be unfolded and assume an open structure optimal
for separation by denaturing EF. Samples of tissues, for example,
are solubilized by rapid homogenization in the solubilizing
solution, after which the sample is centrifuged to pellet insoluble
material and DNA, and the supernatant collected for application to
the IEF gel.
[0031] Because of the likelihood that protein cysteine residues
will become oxidized to cysteic acid or recombine and thus
stabilize refolded, not fully denatured protein structures during
the run, it is desirable to chemically derivatize the cysteines
before analysis. This is typically accomplished by alkylation to
yield a less reactive cysteine derivative.
[0032] Use of 2-D Electrophoresis
[0033] Two-dimensional electrophoresis is widely used to separate
from hundreds to thousands of proteins in a single analysis, in
order to visualize and quantitate the protein composition of
biological samples such as blood plasma, tissues, cultured cells,
etc. The technique was introduced in 1975 by O'Farrell, and has
been used since then in various forms in many laboratories.
[0034] The gel systems known in the art or referenced above,
however, do not provide an integrated, fully automated,
high-throughput system for two-dimensional electrophoresis of
proteins. Moreover, current IPG and slab gel systems are not fully
automated, wherein all operations including gel casting,
processing, sample loading, running and final disposition are
carried out by an integrated, fully automated system. Current gel
systems cannot be fully controlled by a computer and cannot
systematically vary gel, process, sample load and run parameters,
provide positive sample identification, and cannot collect process
data with the object-of optimizing the reproducibility and
resolution of the protein separations.
OBJECT OF THE INVENTION
[0035] It is an object of the present invention to provide means
for fully automated, high throughput two-dimensional
electrophoresis of proteins.
[0036] It is a further object of the present invention to provide a
means of alkylating protein sulfhydryl groups in an automated
manner.
[0037] It is a further object of the present invention to provide
an IPG gel system optimized for use in a two-dimensional gel system
wherein all operations including gel casting, processing, sample
loading, running and final disposition (either by staining for
protein detection or application to a second dimension slab gel for
use in a two-dimensional protein separation) are carried out by an
automated system.
[0038] It is a further object of the present invention to provide
an IPG gel which is not restricted to a planar geometry, but which
instead can be given any cross-section judged optimal for producing
a high-resolution protein separation, and can incorporate varying
cross-sections along its length as required.
[0039] It is a further object of the present invention to provide
an IPG gel strip system that can be fully controlled by a computer,
thereby affording the opportunity to systematically vary gel,
process, sample load and run parameters and collect process data
with the object of optimizing the reproducibility and resolution of
the separation.
[0040] It is a further object of the present invention to provide a
system for SDS slab gel electrophoresis offering facile automation
(the slab gels should be easily handled in a robotic manner during
casting, loading and running).
[0041] It is a further object of the present invention to provide
accurate placement of the sample with respect to the plane of the
slab gel, so as to avoid migration of sample macromolecules in a
distribution that is asymmetric with respect to the plane of the
slab gel, i.e., along one surface.
[0042] It is a further object of the present invention to provide
effective and even cooling of the slab gel surface so that voltage
(and hence heat generated) can be increased, with attendant
improvements in gel resolution (due to shorted run times, and
consequently decreased diffusion time).
[0043] It is a further object of the invention to provide facile
automation of slab gel staining and scanning.
[0044] It is a further object of the invention to provide automated
means for the recovery of selected protein spots or gel zones for
the purpose of protein identification and characterization by means
such as microchemical sequencing or mass spectrometry.
SUMMARY OF THE INVENTION
[0045] The present invention provides an integrated, fully
automated, high-throughput system for two-dimensional
electrophoresis comprised of gel-making machines, gel processing
machines, gel compositions and geometries, gel handling systems,
sample preparation systems, software and methods. The system is
capable of continuous operation at high-throughput, to allow
construction of large quantitative data sets.
[0046] Sample Preparation
[0047] Automated means are provided for treatment of
protein-containing samples to effect the reduction and alkylation
of cysteine sulfhydryl groups contained therein, with the object of
preventing protein loss in the 2-D process through protein
aggregation or refolding associated with sulfhydryl re-oxidation
during the run.
[0048] IEF
[0049] IPG gels are cast in a computer-controlled mold system
capable of repeatedly casting a gel on a film support, advancing
the support, cutting off the strip of support carrying the fresh
gel, and presenting the strip to a robotic arm. The robotic arm
subsequently carries the IPG strip and inserts it in a sequence of
processing stations that implement steps required to prepare the
IPG and use it, including washing, drying, rehydration, sample
loading, and subjection to high voltage.
[0050] The approach used in casting the IPG gel allows the shape of
the gel to depart from conventional flat planar strip geometry. The
method of sample loading allows the sample to be applied over a
large area of the gel. Such a gel format can provide an improved
two-stage separation system: a first stage in which the proteins
are separated in a minimally-restrictive, ideally fluid medium by
isoelectric focusing in a channel or surface layer containing
conventional ampholytes but surrounded by an IPG gel that
establishes the pH gradient, and continuing on to a second stage in
which the proteins are imbibed by the surrounding IPG gel at, or
near their isoelectric points and maintained in stable, focused
positions until the end of the run.
[0051] SDS Slabs
[0052] SDS slab gels used for the second dimension separation are
formed in an automated mold which plays the role of the gel-forming
cassette of a conventional system. By using an approach analogous
to injection molding, the gel is no longer required to assume a
homogeneous planar configuration. In effect, a three-phase gel may
be constructed, having regions corresponding to the separating gel,
stacking gel and upper buffer reservoirs of a conventional slab gel
system. Polymerizable gel solutions can be fed to the mold by one
or more computer-controlled pumping devices, thus facilitating the
creation of multiple zones of gel having different electrochemical
properties. An upper electrode in the form of a rigid bar is
polymerized into one region of the slab gel, allowing it to be
manipulated and transported "bare" (i.e., without any surface
protection or coating) by a second robotic arm (i.e., no
cassette).
[0053] A slot or other means is provided for introducing a sample
(usually in the form of a first dimension gel rod or strip) into or
onto the slab. The slab is "run" (voltage applied) while it is
hanging in a bath of cooled, circulating insulating liquid, such as
silicone oil. The oil prevents evaporation of water from the planar
gel surfaces as the gel runs (a function typically performed by the
glass plates of a conventional gel cassette) and prevents joule
heat caused by the electrophoresis current from raising the
temperature of the gel appreciably. The gel contacts a layer of
aqueous solvent underlying the oil, serving as a lower buffer (with
suitable electrodes). The low density of the oil keeps it above and
unmixed with the lower aqueous buffer.
[0054] After the run, the slab gel is carried by the second robotic
arm to a succession of tanks containing a series of solutions
needed to effect staining of the protein spots or bands on the gel.
Because of differences in the physical densities of these
solutions, the staining can make use of the fact that, as solutes
are exchanged between the hanging gel slab and the solution, a
lamina forms at the surfaces of the slab gel that has a density
different from that of the bulk solvent. Because of this
difference, the fluid in this lamina either rises or falls as a
curtain along the slab surface, and is replaced by fresh solvent.
Hence, depleted solution accumulates at either the top or bottom of
the tank, where it can be removed and replaced with fresh solution.
After staining, the gel can be transported by the robotic arm to a
scanner where it can be digitized for computer analysis.
[0055] Software
[0056] The entire process can be controlled by a computer running
software that both drives the creation and processing of each gel
and collects process data from sensors placed at strategic points
in the production line so as to allow quality control and
optimization. A scheduling algorithm is implemented in software so
that each sample can be run with different gel parameters, if
desired, while ensuring that the manifold actions required to
process one sample do not interfere with actions required to
process other gels in the system (e.g., so that the arm used to
transport IPG gels between processing stations is not required to
be in two places at once).
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 is a schematic diagram of the entire automated 2-D
electrophoresis process.
[0058] FIGS. 2A through 2H illustrate sample preparation using a
size exclusion column.
[0059] FIG. 3 illustrates an IPG gradient maker and mold
system.
[0060] FIGS. 4A and 4B are schematic cross-sections through an IPG
mold system.
[0061] FIGS. 5A through 5F shows a series of six alternative
cross-sections for IPG gels formed by various mold activities.
[0062] FIG. 6 is a schematic view of an IPG strip in a horizontal
position with the gel-side on top of a base plate in position for
sample loading.
[0063] FIG. 7 is a side view of an IPG carrier arm and an IPG slot
run.
[0064] FIGS. 8A through 8E illustrate the sequence of actions of a
slab gel mold during casting operation.
[0065] FIGS. 9A through 9K illustrate alternative forms of slab
gels.
[0066] FIG. 10 is an end view of slab gel run tanks.
[0067] FIG. 11A is an end view of slab gel staining tanks with the
slab gel carrier arm,
[0068] FIG. 11B is an end view of the a slab gel in the holder,
and
[0069] FIG. 11C is an end view of a slab gel held by a clamp.
[0070] FIG. 12 illustrates the placement of a slab gel on a
scanning platform by a slab carrier arm and configuration of
fluorescence illumination.
[0071] FIG. 13 illustrates the sequence of actions of a spot
excision punch.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0072] The preferred embodiment of the automated system for 2-D
electrophoresis described is a continuously-operating production
line, making each gel (both IEF and SDS) just as needed, and
capable of undertaking all steps of the process (FIG. 1)--from
loading of sample onto the first dimension gel to final entry of
protein quantitation data into a computer database.
[0073] Sample Preparation
[0074] In order that proteins retain constant chemical properties
during the process of separation by IPG-IEF and SDS
electrophoresis, it is important that the sulflhydryl (SH) groups
of the cysteine residues that they contain not be allowed to reform
disulfide bridges or become oxidized to cysteic acid during the
separation process. In the preferred embodiment, the protein
cysteine residues are permanently rendered stable by alkylation
with iodoacetamide or one of its uncharged or zwitterionic
derivatives (such as S+2-amino-5-iodoacetamido-pentanoic acid),
which introduces a very hydrophilic group at every cysteine
position but does not change the protein's net charge or apparent
isoelectric point and has a negligible effect on protein mass. This
derivatization is implemented in an automated fashion using a size
exclusion gel filtration column to exchange the proteins out of the
initial sample solubilization solution, through a reagent zone
containing alkylating reagent, and finally into a medium suitable
for application to an IPG gel. The size exclusion media is chosen
so as to exclude proteins but not low molecular weight solvents
(e.g., polyacrylamide beads such as BioRad P-6 BioGel). In
practice, a sample containing a sulflhydryl reducing reagent such
as DTT is removed from a vial selected by a conventional
autosampler such as is used in high performance liquid
chromatography (HPLC), directed by a valve at the head of the
column onto a column which has been pre-equilibrated with the final
sample solvent and a zone (immediately preceding the sample)
containing alkylating reagent in sufficient excess to ensure rapid
reaction with protein cysteines. Once the sample zone is loaded,
the valve switches to deliver a stream of final sample solvent that
propels all the zones down the column and prepares the column for
the succeeding cycle. As the initial sample zone moves down the
column the protein molecules, because of their greater size, fail
to penetrate into the particles of the column packing and hence
move forward at a greater speed than that of the bulk solvent,
which freely exchanges into the volume of the porous particles.
This principle of separation is well known in the art. The proteins
thus move into the zone of alkylating reactant, react there, and
finally move even farther forward into the preceding zone of final
sample solvent. This procedure thus ensures alkylation of protein
sulfflydryls and removal of any low molecular weight contaminants
as well. The sample is then ready for application to an IPG
gel.
[0075] FIG. 2 illustrates a sample preparation apparatus which uses
a size exclusion column. FIG. 2A depicts the arrangement of the
components of the sample preparation apparatus. A size exclusion
column 1 is connected to one of a series of input liquid streams 2,
3 and 4 by a multi-position switching valve 5, with liquid flow
into the column driven by pump 6. Initially column 1 is
equilibrated with liquid 4. Input 2 delivers the crude sample from
a conventional autosampler or other device. Input 3 delivers a
stream of reagent required to effect a chemical treatment of the
sample proteins (typically a sulflhydryl alkylating reagent), and
input 4 delivers a stream of column eluent (the solvent in which
the sample proteins will ultimately emerge). Liquid emerging from
the size exclusion column flows through a UV absorbance or other
column monitor flow cell 7 and thereby to a multiport valve 8 that
directs the eluent either to waste 9 or to a sample collection
vessel 10.
[0076] FIGS. 2B through 2H depict steps in the operation of the
column to effect sample protein derivatization. In FIG. 2B, the
column 1 is equilibrated with eluent through connection of its
input to eluent reservoir 4 by input valve 5, and its output to
waste 9 by output valve 8. Pump 6 and UV monitor 7 are not shown
for clarity: the pump is assumed to remain on during the sequence
of operations, delivering liquid continuously through the column.
In FIG. 2C, a zone of alkylation reagent 11 is introduced onto the
column by switching the input valve 5 to draw solvent from the
alkylation reagent reservoir 3. In FIG. 2D, a zone of sample is
introduced after the alkylating reagent zone, said sample zone
comprising a solvent phase 12 and a protein solute phase 13. In
FIG. 2E, input to the column once again switches to eluent, pushing
the sample and alkylation zones down the column.
[0077] As the sample solvent zone moves down the column, the
proteins it initially contained are excluded from the matrix of the
size exclusion column and hence advance into the alkylation zone (a
well known feature of such columns when used in desalting
applications). During this period, the proteins are exposed to the
alkylating reagents and their component sulfhydryl groups are
alkylated to prevent re-folding of the proteins in subsequent
stages of the 2D electrophoresis process. In FIG. 2F, the proteins
in solute phase continue to advance down the column faster than the
proteins in solvent phase, and enter the leading region comprised
of the first applied eluent phase. In FIG. 2G, the alkylated
proteins are collected by switching the output collection valve 8
to the sample collection position. In FIG. 2H, continuing flow of
eluent into the column forces the alkylation and initial sample
solvent phases out of the column in preparation for the column's
regeneration and re-use.
[0078] In an alternative embodiment, alkylation is performed with a
negatively charged reagent such as iodoacetic acid, thereby
substituting a negative charge at every alkylated protein
sulfhydryl. When this reaction is accomplished stoichiometrically,
very basic proteins containing cysteine residues are shifted
towards more neutral isoelectric points, thereby facilitating their
detection on IEF gels.
[0079] IPG
[0080] The first operation of the 2D gel procedure is creation of
an isoelectric focusing gel to effect the first dimension
separation. Such a separation is most effectively carried out in an
immobilized pH gradient (IPG) gel, in which a gradient of
polymerizable monomers is gelled to form a fixed spatial pH
gradient.
[0081] Gradient
[0082] The compositional gradient required to form the desired pH
gradient IPG gel can be produced by a system of four
computer-controlled motorized syringes delivering, respectively,
heavy gel monomer composition formulated to yield a basic pH, light
gel monomer composition formulated to yield an acidic pH, a
polymerization initiator such as ammonium persulfate, and a
polymerization catalyst such as TEMED. A computer program
constructed, for example, in the LabVIEW language, is used in
conjunction with a computer and stepper motor control card (for
example, a Compumotor AT6400 card) to produce a varying ratio
between the speed of delivery of heavy and light components, while
maintaining a continuous delivery of initiator and catalyst
required for polymerization. Each of the four syringes is connected
to a separate computer-controlled valve (e.g., a 6-port high
pressure liquid chromatography valve in which each of two
rotational positions connects a fixed input with one of two lines
and a fixed output with one of two other lines) that allows
connection of the syringe either to an external reservoir, or to
the delivery tubing system.
[0083] When the syringe is connected to the reservoir for
refilling, the delivery system is connected to a source of
pressurized flush solvent (typically water) that displaces
polymerizable monomer solutions from the delivery tubes to prevent
blockage. In the delivery tubing system, the four component flows
emerging from the four valves are combined by appropriate tubing
junctions to yield one mixed fluid stream routed into the gradient
delivery tube in the mold.
[0084] An additional fifth syringe may be added to supply a third
polymerizable monomer solution of density and pH intermediate
between the light and heavy monomer solutions, for the purpose of
creating very wide pH gradients as a sequence of two two-component
gradients (i.e., A.fwdarw.B followed by B.fwdarw.C).
[0085] FIG. 3 schematically depicts the components of an IPG
casting system. A vertically-oriented mold cavity is formed of a
front mold half 14 and a back surface comprised of activated
Gelbond.RTM. sheet 15. At each casting cycle, fresh Gelbond.RTM. is
delivered to the mold from a roll 16 through motorized transport
rollers 17. A small diameter rigid delivery tube 18 extends into
the mold from the top and may be raised out of the mold by linear
motion 19. A flexible tube 20 delivers a polymerizable composition
to the delivery tube from a gradient maker having five computer
controlled syringes. Each syringe 21 is connected to the output
manifold 22 through a 6-port valve 23 allowing the syringe to be
connected either to a liquid reservoir (e.g., liquid reservoir 24)
for refilling or to the output manifold. These syringes deliver one
of three acrylamide monomer solutions (24, 25, 26) ammonium
persulfate 27 and TEMED 28. Valves attached to syringes drawing
from reservoirs 24, 26, 27 and 28 are shown in delivery position,
while the valve attached to the syringe drawing from reservoir 25
is shown in refill position.
[0086] Each syringe is driven by a motor 29 rotating a lead screw
30 that generates linear motion of a block 31 attached to the
syringe's plunger 32. During the refilling of syringe 21 from its
associated reservoir 24, the associated 6-port valve 23 connects
the output manifold 22 to a pressurized source of non-polymerizable
solvent 33 (e.g., water), to purge the manifold and delivery tubes
of polymerizable media (this configuration shown for the middle
syringe connected to reservoir 25). After delivering a gradient of
polyrnerizable monomers to the mold, the delivery tube 18 is raised
by delivery tube motion 19 so that its open end lies in a block 34
through which air is sucked at high velocity by an air pump, from
input 35 to output 36. A second linear motion 37 carries a long
straight pin 38 which can be inserted into the mold along its axis
or raised out of it.
[0087] The resulting compositional gradient must be delivered into
a suitable mold such that a spatial gradient is maintained during
gelation. In order to achieve this, the delivery tube delivering
gel composition to the mold is arranged on a vertical linear
transport capable of inserting the open end of the delivery-tube to
the bottom of the vertical mold cavity, and raising it slowly as
the gradient is dispensed so as to deposit successive elements of
the gradient above one another (at the rising meniscus of the
liquid in the mold). When the gradient is thus completed, the
delivery tube is raised fully out of the mold and into a suction
block 34 mounted just above the gel mold. In this position, liquid
emerging from the delivery-tube is sucked into a perpendicular
waste tube by the action of a vacuum, thereby providing a waste
path for flush solvents directed through the delivery-tube between
gradient dispensing operations in order to prevent blockage of the
tube by any remaining polymerizable components,
[0088] Suitable compositions for the four components combined to
make an IPG are as follows. Solutions of polymerization catalyst
and initiator (assuming that each comprises 10% of the total volume
dispensed) are, respectively, 1.2% tetramethylethylenediamine
(TEMED) and 1.2% ammonium persulfate (AP), both in water. The two
solutions of polymerizable monomers (whose proportions in the
output stream vary to yield a gradient of titratable monomers and
physical density) may be made up as shown in the following Table to
achieve a gradient over a range of pH 4 to 9. The titratable
monomers used are Immobilines.RTM. manufactured by Pharmacia
Biotech. Glycerol and deuterium oxide (heavy water) are used to
increase the density of one of the solutions, thus helping to
stabilize the gradient formed in the mold through the interaction
of the resulting density gradient and the earth's gravity.
1 TABLE 1 Heavy Light (pH 4) (pH 9) Immobiline pK 3.6 2,762 491
microliters Immobiline pK 4.6 785 1,414 microliters Immobiline pK
6.2 773 1,200 microliters Immobiline pK 7 75 988 microliters
Immobiline pK 8.5 834 236 microliters Immobiline pK 9.3 738 2,209
microliters 31.8% T, 5.6% C Acrylamide/bis in H.sub.2O 0 9.83 ml
31.8% T, 5.6% C Acrylamide/bis in D.sub.2O 9.83 0 ml Glycerol 6.25
0 ml D.sub.2O (Heavy Water) 19.79 0 ml Water 0 25.46 ml 1M Tris
HCl, pH 7.0 8.17 8.17 ml Total 50.00 50.00 ml
[0089] Because the volume of the tubing connecting the gradient
maker with the mold is a significant fraction of the mold volume
(even when narrow-bore HPLC tubing and connectors of inside
diameter 0.010" are used), it is necessary to take account of this
volume when dispensing a gradient. Hence, the procedure adopted and
implemented in the control software consists of five stages: 1)
delivery of the first segment of the desired gradient, equal in
volume to the volume of the delivery tube, for the purpose of
replacing the flush solvent in the tube with polymerizable monomer;
2) insertion of the delivery tube into the mold; 3) delivery of the
remainder of the gradient while the delivery tube is raised
(withdrawn from the mold) at a speed such that the delivered
gradient composition is emitted at the rising surface of the liquid
in the mold; 4) following the gradient by a volume equal to the
delivery tube volume of the final "light" composition, for the
purpose of forcing the section of the gradient remaining in the
delivery tube into the mold while the delivery tube continues to
rise; and 5) removal of the delivery tube from the mold to the
upper vacuum flush position where, following switching of the four
valves, flush liquid is forced through the delivery tube system to
remove polymerizable material and to prepare the system for a
subsequent gradient delivery.
[0090] Mold
[0091] In the preferred embodiment, the IPG is cast in a narrow
vertical mold cavity formed by pressing a movable mold half against
a sheet of Gelbond.RTM. PAG-activated plastic substrate which in
turn is pressed against a fixed backing block whose temperature is
controlled by circulation of chilled or heated water through
internal cavities. The cavity in the movable mold half is
surrounded on the sides and bottom by an O-ring groove with an
O-ring to produce a liquid-tight seal against the Gelbond.RTM.. The
Gelbond.RTM. substrate is made of Mylar.RTM. polyester plastic film
treated in such a way as to produce on its active surface groups to
which an acrylamide gel can bond covalently, thus attaching the gel
to the Gelbond.RTM. substrate.
[0092] In the preferred embodiment, a longitudinal IPG gradient is
formed in the cavity by dispensing a varying composition of gelable
monomers into the cavity through a small diameter delivery tube.
This delivery tube rises during the dispensing of the gradient, and
consequent filling of the mold, so that the open end of the tube
from which gelable monomer emerges is maintained at the rising
level of the surface of the liquid dispensed into the mold. In
addition, the gradient of gelable monomers is contrived so as to
incorporate a physical density gradient that evolves from heavy to
light during the dispensing of the gradient. Such a density
gradient is produced by inclusion of a dense substance such as
glycerol or deuterium oxide in place of a portion of the water
present in the "heavy" gradient component. A density gradient
dispensed in the "heavy" to "light" sequence from a tube maintained
at the rising surface of liquid in the mold gives rise to a stable
composition gradient in the mold which, when polymerized, yields an
IPG.
[0093] FIG. 4 is a schematic cross-section of an IPG mold system
viewed from above (i.e., looking down into the mold cavity 14
depicted in FIG. 3). In FIG. 4A, the front IPG mold half 14 is
pressed against the Gelbond.RTM. sheet 15 by a pneumatic cylinder
39, whose pressure bears on a fixed back plate 40. In this example,
the IPG mold cavity is of a semi-circular cross section 41. Lateral
leakage of polymerizable components is prevented by linear O-rings
42. Mold temperature can be controlled by circulation of hot or
cooled liquid through internal channels of the fixed back plate.
Polymerizable components are introduced into the mold through
moving delivery tube 18. A central hole may be formed in the IPG
gel by polymerization with a pin 38 in place inside the mold
cavity.
[0094] In FIG. 4B, following extraction of the delivery tube and
pin from the mold, pneumatic cylinder 39 retracts the front IPG
mold half 14, and rollers 17 cause the Gelbond.RTM. support 15 to
slide laterally across the face of fixed back plate 40, thereby
ejecting the IPG gel 43 on its Gelbond.RTM. substrate from the
mold, in preparation for another cycle. A rotary blade 44 cuts the
Gelbond.RTM. by moving vertically along the mold, thereby releasing
a strip of Gelbond.RTM. carrying the newly-formed IPG gel. The gel
produced has a longitudinal hole 45.
[0095] It is important to ensure that the gradient of the resulting
gel reaches hydrostatic equilibrium (and hence proper gradient
shape) before polymerization, and yet is fully polymerized (with
complete incorporation of gelable monomers into the gel polymer
matrix) before removal from the mold. This result is achieved by
increasing the temperature in the mold after an initial gradient
formation period: gelation proceeds much faster at higher
temperature. In a typical protocol, the gel gradient is introduced
at a temperature of 20.degree. C. and after a period of
approximately 4 minutes, during which the polymerizable monomers
gel into a non-convecting state, the temperature in the mold is
increased to approximately 50.degree. C. by circulation of heated
water through closed channels provided in the backing plate. After
removal of the gel from the mold, the temperature is lowered to
20.degree. C. by switching the circulation system to a chilled
water supply in preparation for the next cycle.
[0096] Once the gel is polymerized, the mold is opened and the IPG
gel is transported by manipulation of the Gelbond.RTM. support to
which it has become covalently attached during polymerization. The
form of the gel is determined by the form of the mold in which it
is cast, the simplest being a flat, rectangular strip on the
surface of the Gelbond.RTM..
[0097] In a further embodiment, the gradient stream of
polymerizable monomers is introduced into the mold cavity by means
of a passage at the bottom of the cavity, in this case in the
sequence light to heavy (opposite to the order when liquid is
deposited at the rising surface of the liquid in the mold). A
special valve is used to direct the flow of polymerizable liquid
either into the mold or to waste, thereby allowing the contents of
the delivery tubing to be purged of polymerizable components after
casting of a gel.
[0098] Numerous alternative forms of the IPG gel can be produced.
In one alternative embodiment, a pin is introduced into the mold
before or during gel polymerization and slowly withdrawn
afterwards, leaving a central hole down the length of the IPG gel.
This can be accomplished by a procedure in which the pin is first
rotated slowly, to reduce the adhesion of gel to the pin, and
subsequently slowly withdrawn along its axis through the top of the
mold. In another embodiment, a sample introduction channel or
groove is formed at the exterior surface of the IPG gel by means of
a suitably shaped ridge on the interior surface of the mold. The
groove may be formed so as to be closed at its ends, thus forming a
bounded depression, open only at the top. Provided that the gel is
held horizontal during the run, i.e. with the groove in a
horizontal plane and with its opening directed upward or to the
side, then sample liquid placed in this groove will remain held
there by capillary action, until imbibed by the gel or
evaporated.
[0099] FIG. 5 shows a series of six alternative cross-sections for
IPG gels formed by various mold cavities, after the strip has been
cut from the Gelbond.RTM. roll. In FIG. 5A, a semi-circular gel 43
with longitudinal hole 45 has been formed on the Gelbond.RTM. strip
46 and subsequently filled with sample. In FIG. 5B, a semi-circular
cross-section gel has a surface groove 47 in which sample is held
by capillary action, while in FIGS. 5C and 5D, other cross-sections
with broader, flatter surface grooves 47 are shown, also holding
sample by capillarity. In FIGS. 5E and 5F, triangular and
rectangular cross-sections without surface grooves are shown. In
each case shown, the Gelbond.RTM. backing material is wider than
the gel itself, giving the strip greater stiffness and providing,
particularly in FIGS. 5E and 5F, a further form of cavity in which
sample is held by capillarity: a groove created by the included
angle between the side of the gel on one hand and the extended
Gelbond.RTM. substrate on the other.
[0100] In practice, the gel mold can be formed from any of a range
of materials that do not inhibit polymerization of acrylamide,
including glass, alumina, machinable ceramic, Ultem.RTM.,
polysulfone, polystyrene, polycarbonate, polyurethane, acrylic,
polyethylene or the like. For convenience in machining, and to
allow observation of the mold's contents, a clear plastic such as
polysulfone or acrylic is preferred.
[0101] Gelbond.RTM. Transport
[0102] Gelbond.RTM. substrate is advanced to the mold on repeated
cycles from a large roll by feed rollers. After casting an IPG gel
on the end of the Gelbond.RTM. (the IPG axis perpendicular to the
length of the Gelbond.RTM. and parallel to the roll's axis), the
strip of Gelbond.RTM. on which the gel is formed is cut from the
roll using any of a variety of mechanical cutting mechanisms,
including, for example, a rolling disk cutter of the type used to
cut photographic paper, affixed to a vertical motion device. The
resulting Gelbond.RTM. strip with IPG gel attached may then be
grasped by any of a variety of mechanical or manual means for
handling in further processing steps. In the preferred embodiment,
the strip is 1.27 cm wide and approximately 65 cm long (the width
of the Gelbond.RTM. substrate when provided in roll form). The IPG
gel is 2 mm wide, 0.75 mm thick and 57 cm long (leaving 1.5 cm of
the Gelbond.RTM. uncovered on either end of the strip).
[0103] Barcode Labeler
[0104] Preprinted barcoded labels are mechanically applied to each
IPG-carrying strip on the side opposite the gel for identification
purposes, although other labeling means known or available may be
used.
[0105] Robotic Arm
[0106] A robotic arm system equipped with two
pneumatically-activated pincers grasps the strip by the two ends to
transport it between subsequent processing stations. The IPG arm
system moves horizontally along a track, vertically along a linear
table mounted on the track, and can rotate 90 degrees in order to
pick up the IPG lying in a horizontal position and carry it in a
vertical orientation to subsequent stations.
[0107] Sequence of IPG Processing Events
[0108] To employ a gel made by the procedure described above as the
first dimension separation of a 2-D electrophoresis procedure, a
sequence of processing operations, many of which have been well
described in the art, is used to render the gel ready for use in a
protein separation. These operations include removal of remaining
unpolymerized monomers, initiator and catalyst by washing in
deionized water; dehydration to remove incorporated water; and
finally rehydration in a solution appropriate as a medium for
protein separation. Subsequently, a protein-containing sample is
applied to the gel, and the gel is subjected to a voltage gradient
in order to separate the proteins along the gel length.
[0109] In the preferred embodiment, the IPG gel on its Gelbond.RTM.
strip is gripped at both ends by the aforementioned movable arm and
placed in one of a plurality of slots containing circulating
purified water. After approximately two hours, most soluble
materials remaining in the gel have diffused into the water and are
thus removed from the gel.
[0110] The strip is then grasped again by the arm (which in the
meantime may have moved to other positions to carry out other
functions) and moved to a slot where it is subjected to a stream of
air filtered so as to remove any contaminating particulate material
(e.g., using a conventional HEPA filter). The gel is substantially
dried in approximately 30 minutes.
[0111] Next, the arm again grasps the strip and moves it to a slot
filled with rehydration solution, a medium typically consisting of
9 mole/liter urea, 2% of a non-ionic detergent such as Nonidet P-40
or CHAPS, and 2% wide range, commercially-available ampholytes
(e.g., BDH 3-10 ampholytes) in water. When samples are to be used
whose protein SH groups have not been alkylated, 1% dithiothreitol
is included in the rehydration solution as a sulfhydryl reducing
agent. In a period of approximately two hours, the IPG gel is
re-swollen in rehydration solution and ready to be used for protein
separation. In order to prevent the formation of crystals due to
evaporation at the surface of the rehydration solution bath, the
rehydration solution is covered by a layer of light silicone oil,
through which the IPG is inserted.
[0112] To carry out a protein separation, a volume of sample
protein must be applied to the gel. In the preferred embodiment,
sample protein in a solubilization solution similar in composition
to the rehydration solution is applied on the surface of the IPG
gel along its length. This application is effected by placing the
IPG on a base plate with the gel face up, and depositing a stream
of sample liquid onto the IPG gel surface from a needle held just
above that surface, which is moved slowly along the length of the
IPG as sample is pumped out. The resulting thin layer of
protein-containing liquid on the IPG gel surface remains in place
during subsequent manipulations of the gel strip so long as the
axis of the gel remains in a horizontal plane (as is the case
during movement using the arm system described). Means are provided
for moving the needle up and down (to allow collection of sample by
piercing the septum of a conventional septum-topped sample vial),
and for moving it along the length of the IPG and farther, to
positions where a sample vial may be placed and where the needle
may be washed.
[0113] FIG. 6 shows an apparatus for application of sample protein
to an isoelectric focusing gel in accordance with the present
invention. An IPG strip 46 lies horizontally, gel-side up, on a
base plate 48. A trail of sample liquid 49 is left on the surface
of the IPG gel as needle 50 discharges a steady stream of sample
while moving along the IPG. The needle 50 is moved on carriage 51
through the action of lead screw 52 driven by motor 53. Sample flow
is controlled by syringe 54 whose plunger 55 is moved by a block 56
which is in turn moved by a lead screw 57 turned by motor 58.
Flexible tube 59 connects the syringe and the delivery needle. The
sample is initially taken into the needle 50 and syringe 54 by
raising the needle on its vertical pneumatic motion 60, driving the
needle 50 to the left, positioning it over sample vial 61, lowering
the needle 50 to pierce the vial's septum top, withdrawing the
sample through action of the syringe 54, raising the needle 50
again, moving into position over a gel strip, lowering the needle
50 and commencing synchronous motion of the syringe 54 and the
needle carriage 51 to deposit the sample along the IPG surface. The
needle 50 is washed between applications by positioning it over
waste receptacle 62, where its exterior surface is washed by a jet
of water 63.
[0114] In another embodiment, where a central hole is produced in
the IPG gel during casting, the sample can be injected or
peristaltically drawn into the channel prior to application of
voltage along the gel. The sample liquid can be retained inside the
channel by pinching the ends of the gel to close the channel, by
injection of gas bubbles, or by various other means, including
placing a drop of gelling material at both ends.
[0115] After sample loading, the gel strip is once again grasped by
the arm and moved to one of a plurality of slots tilled with a
non-conducting oil (such as silicone oil) and having slotted carbon
electrodes at either end positioned so as to contact the ends of
the IPG gel. The oil may be circulated, cooled to ensure constant
running temperature and sparged with a dry gas so as to eliminate
oxygen and dissolved water. Since the resistance of the IPG gel
rises during the run, slots maintained at a series of different
voltages are provided, and the arm periodically moves the strip
from one voltage to a higher voltage as the run progresses. In the
preferred embodiment, a series of 6 voltage stages are provided,
namely 1, 2.5, 5, 10, 20 and 40 kilovolts. The gel is maintained at
each voltage for about 3 hours, except the last, where it rests
until a second dimension slab gel is available. A total of 200,000
to 300,000 volt-hours may applied to each gel.
[0116] Slots such as those used for washing and for subsequent
processing and running steps generally have clips at either end
into which the gel strip is inserted by the arm, using a downward
motion. When the grasping pincers at the ends of the arm release
the Gelbond.RTM. strip, these clips continue to hold the strip
extended between them by friction. In the preferred embodiment,
these clips consist of a pair of parallel pins touching one another
and projecting upwards from the floor of the slot. The strip is
jammed between these pins during insertion into the slot, spreading
them slightly and producing a friction fit. All the slots except
the air dryer are contained at the sides and below to yield a
liquid-tight vessel suitable for containing the liquid with which
the IPG is to be treated at that stage. Slots used for application
of high voltage also contain slotted carbon electrodes.
[0117] FIG. 7 shows a cross-section view of an IPG processing slot
and the arm used to transport IPG strips between slots. A
Gelbond.RTM. strip 46 carrying attached IPG gel 43 is held at its
ends 64 and 65 by a distal arm 70 and a proximal arm 68, each
carrying a gripper 66 actuated by a pneumatic cylinder 67. Both
arms are mounted on a horizontal bar 69. One of the arms, in this
case the distal arm 70, is mounted to a carriage 71 capable of
moving along bar 69 under the control of belt 72, which in turn is
moved by motor and pulley 73. Since the other arm 68 is fixed to
the horizontal bar 69, movement of arm 70 by the motor and pulley
in an outwards direction serves to stretch strip 46, keeping it
taut (and therefore straight) between grippers 66. A vertical
motion 74 serves to raise and lower the entire arm and bar
assembly, thus allowing insertion of IPG gels into, and removal of
gels from, the slots. The vertical motion is itself carried on
motor-driven wheels 75 which engage a track 76 to move the arm
assembly to positions over a variety of slots.
[0118] Movement of the arm assembly downwards (by motion of
vertical motion 74) causes gel strip 46 and attached IPG gel 43 to
be inserted into a processing slot in plate 77. The strip is held
at its ends between pairs of pins 78 projecting from the floor of
the slot, and is inserted beneath the surface of liquid 79. This
liquid can be circulated over the IPG strip by introducing liquid
through inlet 80 and simultaneously withdrawing liquid through
outlet 81. Excess liquid flows over a dam 82 to exit via overflow
83. In slots devoted to the IEF process (where voltage is applied
across the gel) the ends of the IPG gel 43 contact slotted
electrodes 84, which are connected in turn to conducting pins 85
that penetrate the bottoms of the run slots in a liquid-tight
manner, allowing electrical connection to a power supply on the
outside.
[0119] During the early stages of a separation run, under an
applied electric field, proteins can migrate through the liquid
phase of the applied sample along a pH gradient initially formed by
the action of the ampholytes incorporated in the sample. Because
the proteins are initially migrating through liquid, without the
retardation associated with migration through a gel matrix, they
can approach their isoelectric points more rapidly than in a system
where the entire migration path is through IPG gel. However, if
proteins remained in this liquid phase at the end of the run, they
could be displaced from their isoelectric positions by subsequent
gel handling steps. Hence, conditions are contrived so that, as the
run progresses, sample-containing liquid is imbibed by the gel,
progressively shrinking the channel so that at the end of the run
the channel contains a negligible amount of liquid. This is
achieved by allowing surface water to be slowly removed from the
exterior surface of the gel during the run by, for example,
immersion of the gel in circulated silicone oil that has been
dehydrated by sparging with a dry gas such as argon or
nitrogen.
[0120] During gel dehydration, and consequent collapse of any
liquid filled central sample channel, proteins enter the gel at
positions near their respective isoelectric points. Thus, a mixture
of different proteins will enter the gel at points distributed
along the gel length, rather than at one site at the edge of a
sample well, thereby avoiding the precipitation often observed when
a complex mixture of proteins migrates together into the gel
through a small gel surface area. Excess liquid is removed through
the exterior gel surface, either to a dry gas phase or to a
water-extracting, non-aqueous, non-conducting liquid phase such as
silicone oil.
[0121] SDS Electrophoresis
[0122] Slab Gel Casting
[0123] In the preferred embodiment, a gel is formed in a
computer-controlled mold system whose operation is shown
diagrammatically (in cross-section) in FIG. 8. The mold is composed
of two halves 86 and 87 which can be forced together to comprise a
liquid-tight cavity open at the top, The form of the mold is such
that the gel 89 formed therein has a large, thin planar region at
the bottom (within which proteins will be separated: the
"separating gel") and above the thin planar region a substantially
wider region (the "top gel") joined to the thin region by a joining
region of gradually increasing width. The function of the top gel
is to provide a buffer reservoir as a source of ions during the
electrophoresis separation, and a mechanical support from which the
separating gel hangs during the run and subsequent steps. The
joining region joins the separating and top gel regions and
provides a gradually narrowing cross-section adapted for the
focusing of protein zones using the stacking process disclosed in
Laemmli (U.K., 1970, Nature 227, 680), in which the joining region
is comprised of a stacking gel. In the preferred embodiment, the
separating region has a thickness of about 1 mm, the top region has
a thickness of about 2 cm, and the joining region gives rise to a
smooth fillet between the separating and top gels. The vertical
height of the separating gel is 30 cm and that of the top gel is 5
cm. All gel regions have the same width, namely 60 cm.
[0124] Mixtures of polymerizable gel monomers are introduced into
the closed mold by means of three tubes 88, 90 and 95 which can be
made to extend down into the mold cavity from above. The first
delivery tube 88 can be caused to extend to the bottom of the mold
and is used to introduce a liquid stream that polymerizes to yield
the separating gel 89. A second delivery tube 90 can be made to
extend down inside the upper, wider section of the gel mold, and is
used for the introduction of the second gel phase (the stacking gel
91) and (by means of switching a valve) an equilibration solution
used to bathe the IPG applied to the slab gel. A third delivery
tube 95 also can be made to extend into the upper section of the
gel mold, and is used to introduce the liquid that polymerizes into
the top reservoir gel phase.
[0125] A slot form 92 can be lowered into the open top of the mold
cavity by vertical movement of the slot form. The mold can be
opened by means of another movement, whereby one face of the mold
pivots along a line near to and parallel with the bottom horizontal
edge of the mold cavity to expose the gel. The mold cavity contains
indentations at either end shaped so as to receive and support the
ends of a carbon electrode rod 94 and suspend it inside the top gel
volume during its polymerization. After polymerization of the gel,
electrode rod 94 serves as both an upper electrode required for the
electrophoresis separation and a mechanical support from which the
gel hangs during subsequent handling and manipulation. A further
controlled motion is provided to clamp the electrode rod to one
face of the gel mold, thus ensuring that the gel will always be
recovered in a fixed location after the mold is opened.
[0126] FIG. 8 illustrates the sequence of actions of slab gel mold
during the casting operation. In FIG. 8A, a slab gel mold comprised
of a fixed mold half 86 and a movable mold half 87 is shown in the
closed position. A long delivery tube 88 is extended downwards to
the bottom of the mold, and the polymerizable mixture which will
form the separating gel is dispensed. The motions of this tube and
other delivery tubes are controlled by simple vertical
electromechanical movements. In FIG. 9B, after the separating gel
89 is polymerized, a second shorter delivery tube 90 is lowered and
a stacking gel phase is dispensed. In FIG. 9C, before the stacking
gel 91 polymerizes, a slot form 92 is inserted into the mold to
form the sample slot 93. In FIG. 9D, once the stacking gel is
polymerized, the slot form is withdrawn, an electrode rod 94 is
inserted into the mold, and a third delivery tube 95 is lowered
into the mold to dispense a top gel mixture. In FIG. 9E, after the
top gel 96 is polymerized, the mold is opened. Once the mold is
opened, a completed slab gel 97 hanging from the electrode rod 94
is slowly and evenly removed by slab gel handling arm 98 having an
actuated gripper 99. The arm is carried vertically and horizontally
by linear motion components 100 and 101.
[0127] FIGS. 9A through 9K illustrate alternative forms of slab
gels. The preferred form of slab gel shown in FIG. 9A comprises
three gel phases (separating gel 89, stacking gel 91, and top gel
96), an internal slot-shaped cavity 93 to accommodate the IPG first
dimension gel 46, and a rod-shaped electrode 94. In FIG. 9B, the
stacking gel phase is eliminated and the internal slot 93 is formed
directly in the separating gel 89. In FIG. 9C, the sample slot 93
extends to the top gel surface, while two internal electrode rods
94a and 94b are used. In FIG. 9D, the sample slot 93 also extends
to the upper surface, but the electrode rods 102a and 102b are
external to the gel and support it by interacting with lips 103 on
the gel's external surfaces. In FIG. 9E, the IPG gel 46 is applied
to an external face of the stacking gel phase rather than being
placed in an internal slot, remaining in place as a result of
surface tension. In FIG. 9F, the IPG gel 46 is also applied
externally, but to the separating gel 89 (the stacking gel 91
having been eliminated). In FIG. 9G, the top phase 96 of a gel
configured as in FIG. 9E is rotated counterclockwise by
approximately 160 degrees. By rotating the incorporated electrode
rod 94, the top gel phase 96 is brought in contact with the
separating gel 89, bypassing the stacking gel 91 phase and the IPG
gel 46, after sample proteins have entered into the separating
phase.
[0128] A series of alternative embodiments make use of a gel clamp,
instead of a distinct gel region, to provide an electrode and
source of ions. In FIG. 9H, a hinged clamp, comprised of halves 104
and 105, grasps the top edge of a slab gel and holds it as a result
of the closing force exerted by spring 106. One of the two opposing
faces (105) contains an internal cavity 107 and electrode 108, the
cavity forming a liquid-tight vessel when the gel is clamped in
place thereby covering opening 109. The gel is prevented from
slipping out of the clamp by the presence of a region of increased
gel thickness 110 along the top gel edge, in this case including a
molded-in rod 111 as a means of handling the gel before
introduction into the clamp, and secondarily by the presence of a
gritty coating on one or both of the opposing faces of the clamp.
Projections 112 above the clamp's axis 113 can be squeezed together
to open the clamp and release the gel. Axis 113 is connected
electrically to the liquid vessel's electrode. An IPG gel 46 is
applied on the surface of the slab gel. Once the gel is grasped and
the chamber 107 is filled with an appropriate volume of electrode
buffer, the assembly can be grasped in turn by external means via
axis 113, and manipulated by a robot arm as in the case of the gels
with incorporated electrode rods (e.g., FIG. 9A). The electrode
buffer solution provides the source of ions for electrophoresis,
using the axis 113 as a convenient external electrical contact.
[0129] In FIG. 91, a similar clamp is used to grasp a planar slab
gel having no region of increased gel thickness along the top gel
edge. The gel is prevented from slipping out of the clamp only by
the grasping force and the presence of a gritty coating 114 on one
or both opposing faces of the clamp. In FIG. 9J, the IPG gel is
placed within the clamp on a support structure 115, and thereby
held against the slab gel. The buffer-containing internal cavity is
formed to provide two paths of current flow 116 and 117 into the
slab gel: one above and a smaller one below the IPG. This
arrangement provides a means for directing the proteins transported
from a surface-applied IPG during electrophoresis into the center
plane of the slab gel. Hence, instead of moving along the surface
of the slab to which they were applied (in the case where the IPG
is applied to a surface, rather than inside of the slab), the
protein zone is pushed towards the interior of the gel by the flow
of buffer ions entering through the second path 117. In FIG. 9K,
the clamp contains a channel 118 through which buffer can be
circulated. One leg of this channel 118 runs along the top edge of
the slab gel, where one of the channel's walls is comprised of the
gel's surface, and contains an electrode 108. This channel further
communicates through additional passages 119 with an external
buffer circulation system. In this embodiment, buffer is circulated
through the clamp during the run, providing a supply of fresh
buffer components which, with the electrode mounted in the channel,
allow sustained electrophoresis with a minimum volume of
reagents.
[0130] In the preferred embodiment, a separating gel (usually a
gradient composition varying between approximately 18% T acrylamide
at the bottom of the gel mold to 11% T acrylamide at the top of the
separating gel phase) is introduced through the first delivery tube
88 (FIG. 8A) while it is extended to the bottom of the mold cavity.
This gradient is produced by a second gradient maker similar in
structure to that disclosed above to create an IPG gradient, except
that larger syringes are used to produce a total separating gel
volume of approximately 200 ml. After the gel is introduced, the
first delivery tube 88 is raised out of the mold so that its open
end lies in a block with vacuum channels that direct a stream of
air across the end of the tube and thus aspirate emerging liquid
into a waste container. Multiport valves associated with the
gradient maker syringes are switched so that the syringes may be
refilled, and so that a supply of pressurized water is connected
with the manifold leading to the delivery tube, thus purging it of
polymerizable components and flushing it with water. These
techniques for providing and aspirating delivery wash solvent
function in a manner similar to that described above for IPG gel
formation. The separating gel is left undisturbed to polymerize for
approximately 5 minutes.
[0131] After initial polymerization, a second gel phase, a stacking
gel 91, is formed by extending the second delivery tube 90 into the
top of the mold and dispensing approximately 50 ml of polymerizable
stacking gel mixture directly atop the separating gel. The stacking
gel 91 mix is formed by combining the output of three
computer-controlled syringes delivering stacking gel mix, ammonium
persulfate and TEMED. Before this gel phase polymerizes, the slot
form 92 is caused to move down into the top of the slab gel mold.
The slot form 92 consists of a thin strip (.about.1 mm thick) of
plastic mounted so as to present a vertical edge that lies on the
mold center line which extends to within 1 cm of the separating gel
top and within 1 mm of the diverging walls of the mold in the
joining region. The slot form 92 is approximately 58 cm wide,
leaving a 1 cm open space at either of its ends.
[0132] The stacking gel 91 volume is so contrived that the joining
region is filled with stacking gel mixture up to a depth on the
slot form of approximately 3 mm. Upon polymerization of the
stacking gel 91, the slot form 92 thus creates a slot 3 mm deep in
the horn-shaped stacking gel cross-section, into which an IPG gel
46 or other protein containing sample may be placed.
[0133] After polymerization of the stacking gel 91, the slot form
92 is withdrawn from the mold, and the arm system used for IPG
manipulation is used to place an IPG strip in the slot so formed.
Once this arm is again removed from the mold area, the second
delivery tube 90 is once again introduced into the mold, and a
volume of IPG equilibration solution is dispensed through it into
the slot occupied by the IPG. This equilibration solution
(consisting of 10% glycerol, 5 mM DTT, 2% SDS, 0.125M Tris HCl pH
6.8 and a trace of bromophenol blue) serves to infuse SDS into the
IPG gel 46 and alter its pH to that of the stacking gel 91 in
preparation for stacking. The second delivery tube 90 is then once
again removed from the mold.
[0134] A second movable arm system, as shown in FIG. 12, then
carries a carbon electrode rod 94 (or rods 99) to the mold and
positions it within the mold, approximately 1 cm from the top of
the mold cavity. The electrode rod ends rest in indentations at the
ends of the mold cavity, maintaining the rod in position when
released by the arm, which moves away from the mold after
depositing the rod. The third delivery tube 95 is then introduced
into the mold where it dispenses the third gel phase 96 (the top
gel), filling the mold to the top. This top gel phase 96 is
produced by a peristaltic pump system combining four components: an
acrylamide/bis solution, a buffer solution, ammonium persulfate and
TEMED.
[0135] The result is a slab gel in three phases, with the IPG first
dimension gel 46 and a carbon electrode rod 94 polymerized inside.
The polymerizable gel solutions for these three phases are designed
to polymerize rapidly, so that the three phases adhere to one
another and yield an integral gel whose regions have distinct
electrochemical properties.
[0136] Preferred compositions for the three phases are as follows.
Acrylaide.TM. (FMC Corporation) is an alternative gel crosslinker
which may be used to increase gel strength in the stacking gel.
2 Separating gel Acrylamide 13.00% T bis acrylamide 3.8% C Tris HCl
pH 8.6 0.375 M Stacking gel Acrylamide 8.00% T Tris HCl pH 7.0
0.375 M Acrylaide 2% 3.2% C SDS 0.2% Top electrode gel Acrylamide
13.00% T Tris base 0.048 M Glycine 0.4 M SDS 0.20%
[0137] After the gel is made, the mold is opened by moving apart
the mold halves 86 and 87 and leaving the gel on the movable, now
nearly horizontal, mold half 87. A second computer-controlled arm
system, equipped with two graspers or pincers 99 designed to engage
the opposite ends of the electrode rod 94, is moved into position
to seize the electrode rod 94 and then lift the gel upward and out
of the mold. Gravity causes the gel to hang downwards from the
bar.
[0138] Slab Gel Electrophoresis
[0139] The arm is then moved laterally into position over an empty
slot in a slab gel running tank and slowly lowers the slab gel into
the slot. FIG. 10 illustrates a slab gel running tank in accordance
with the present invention, wherein a slab gel 97 is suspended
vertically in silicone oil during the second dimension
electrophoresis run. The slab 97 is suspended by electrode rod 94
which rests on electrical bus bars 120 (one at either end of the
gel), with the slab gel 97 inserted into a vertical slot through
which cooled silicone oil is circulated. The oil circulation path
is so contrived as to cause laminar flow of a curtain of oil
downwards along both surfaces of the slab gel, thereby removing
joule heat generated during electrophoresis. The oil is recovered
at the bottom of the slots and recirculates through an external
pump and heat exchanger, and thereafter is reintroduced into the
top of the slot in a closed-loop system. This curtain-like flow of
oil serves to prevent the slab gel 97 from touching the walls of
the slot, and insulates it from electrical contact along its
length. Oil enters the tank through manifold 121, is distributed to
supply plenums 122, expelled though holes 123 into the gel slot,
and flows down the slot on either side of the separating gel 89, to
be sucked out through return manifold 124 via return plenums 125
and return holes 126.
[0140] At the bottom of the tank, below the level of the bottom of
the slots, a lower electrically-conductive aqueous phase 127
(denser than the silicone oil) is positioned so that it just
contacts the bottom edge of the slab gel 97. Current passes from
the electrode bar or bars embedded in the top gel 96 through the
stacking gel 91 and separating gel 89 to the lower aqueous phase
and lower electrode 128, thus completing the circuit required for
an electrophoretic separation. The shield 129 is provided over the
lower electrode 128 to funnel the bubbles generated there to one
side and up a separate pipe, thus preventing their rising through
the aqueous phase and then the silicone oil phase, and causing
mixing of the two phases.
[0141] At a voltage of 600 volts and a current of 1 amp, the
separation of proteins in the separating gel 97 can be effected in
approximately 4 to 5 hours. Once the separation is complete, the
aforementioned slab gel arm system is used to grasp the ends of the
electrode bar 94, raise the gel out of the running slot and move
the gel into position over the first of several tanks containing
solutions required to visualize the separated proteins by
staining.
[0142] Slab gels and electrophoresis methods of the type disclosed
can be used for separation of samples other than proteins contained
in IPG gels. In particular, the inclusion of multiple sample wells
in place of the single slot provided for an IPG allows use of such
gels to separate protein or nucleic acid components of numerous
liquid samples.
[0143] Slab Gel Staining
[0144] Several stain protocols can be executed including, among
many others, staining with Coomassie Brilliant blue, ammoniacal
silver, silver nitrate, and fluorescent stains such as SYPRO red
and orange. The following example exemplifies the method applied to
any stain. The gel is moved between subsequent tanks, by the arm
under computer control, so that the precise time of movement from
one solution to the next can be controlled, and can be held
generally constant from gel to gel.
[0145] In a first tank, the gel is immersed up to the stacking gel
in a solution of 30% ethanol, 2% phosphoric acid and 68% water for
a period of two hours, to fix the proteins in place and remove most
of the SDS, Tris and glycine in the gel. Following this fixation
step, the gel is moved, through use of the arm, to a tank of 28%
methanol, 14% ammonium sulfate, 2% phosphoric acid in water, where
it is incubated for two hours. Next, the gel is moved to a tank of
the same composition with the addition of powdered Coomassie Blue
G250 dye, the whole liquid volume being continually circulated or
agitated in the tank. Here the dye permeates the gel, binding to
resolved protein spots. Finally, the gel is removed from this tank
and transported by the arm to a scanning station.
[0146] FIG. 11A illustrates slab gel staining tanks with a slab
carrier arm. In order to expose slab gels 97 to staining solutions,
the gels are suspended in staining tanks 130, where they are
supported by the embedded electrode rods 94 whose ends sit on
projecting supports 131. The tank 130 is filled with stain solution
132, which can be removed from the tank by opening exit valve 133.
The tank 130 can be refilled by closing valve 133 and then opening
input valve 134 and activating pump 135 to deliver solution 132
from reservoir 136. Solutions in the tank can be agitated when
required by a variety of means well known in the photographic
processing industry, including bursts of inert gas (such as
nitrogen or argon) introduced at the bottom of the tank, or by
small mechanical motions of the suspended gels caused by cyclic
movement of the gel supports 131. Gels 97 are moved from tank to
tank by means of arm 98 having pneumatically controlled grippers 99
which seize the ends of electrode rod 94. The arm 98 is raised and
lowered by vertical movement 100 which in turn rides on lateral
movement 101, all under computer control.
[0147] FIGS. 11B and 11C show alternative embodiments allowing gels
without incorporated electrode rods to be similarly processed. In
FIG. 11B, a slab gel 89 is contained inside a holder whose two
halves 137 and 138 are connected by hinge 139 at the top edge and
held together by magnets 140 at the bottom edge. Each half of the
rectangular holder has a large cutout and is shaped like a picture
frame. One surface of each half is covered with a taut mesh 141,
resulting in a narrow gel cavity with large-area porous walls. A
slab gel placed in such a holder is thus exposed to any solution
into which the holder is immersed, and can be processed through a
series of tanks using a robot arm to grasp projecting pins 142. In
FIG. 11C, an alternative slab gel holder makes use of a clamp
hinged at 142, held together by magnets 143 and having its internal
faces 144 coated with a gritty coating, to grasp a slab gel for
transportation and processing. Projections 145 may be squeezed
together to open the clamp, releasing the gel.
[0148] Scanning
[0149] In order to obtain quantitative data on the abundance of
resolved proteins, the gel is scanned to yield a digitized image.
FIG. 12 shows a gel 97 being gently laid down on a horizontal or
tilted illuminating table 146 prior to scanning, grasped as before
by the electrode rod 94 embedded in its top phase 96. To do this,
the robotic arm 98 executes a coordinated vertical and horizontal
motion so that the gel is laid down smoothly without tension. An
overhead digital camera 147, such as a CCD digitizer, may then be
used to acquire an image of the gel 97 and its stained protein
spots in absorbance mode. In order to allow scanning of a large
area gel at high resolution, a camera covering, for example,
1024.times.1024 pixels can be moved to a series of locations by
orthogonal linear motions 148 and 149, generating a series of scans
that can be combined to yield a larger image. Alternative scanning
and illumination modes may be provided for measuring fluorescence
or light scattering, in situations where the proteins have been
stained with a fluorescent or a particulate dye, respectively. In
the preferred embodiment, fluorescence excitation illumination is
delivered to the gel in the plane of the gel while it lies in a
horizontal cavity defined by walls 151 and filled with a liquid
152, such as water, having a refractive index similar to the gel.
Light is piped into the cavity by an optical fiber light pipe 153,
one of whose ends pierces the walls 151, the other end being
illuminated by light produced by light source 155 filtered by
interposed optical filter 154. In fluorescence mode, light emitted
by fluorescent moieties in the gel is detected by the digitizer
after passage through a second optical filter 150 which passes the
dye's emission wavelength while blocking the excitation light. The
approach described makes use of the fact that the exciting light is
trapped by internal reflections in the gel/water plane, thus
improving its availability to excite protein-bound fluorescent dye
molecules and diminishing the amount of exciting light that escapes
normal to the gel plane to impinge on the detector. A similar
optical system, but without a requirement for excitation and
emission filters, can be used to detect light scattering by
particles generated either on the protein spots (for example by the
silver stain) or around the spots (leaving the proteins negatively
stained, as occurs with the copper stain).
[0150] Using the automated staining system described, multiple
stain and scan cycles can be sequentially applied to the same gel.
By staining first with a relatively low sensitivity stain such as
Coomassie Blue and scanning, and then staining with a relatively
sensitive stain such as the silver stain and scanning once again,
it is possible to obtain quantitative protein abundance measurement
over a wider dynamic range than can be afforded by any single
conventional stain.
[0151] Multiple sequential scans of the same gel may be used to
increase the precision and dynamic range of non-equilibrium stains
such as the silver stain. In such stains, the development process
reveals first the intensely staining spots (in general the more
abundant proteins), then those of moderate staining intensity, and
finally those of low staining intensity (typically low abundance
proteins), at which point the intensely staining spots are over
stained, being saturated in stain absorbance and appearing
increased in size. By scanning the gel two or more times during
development, quantitation of spots can be based on measurements of
parameters other than simple optical density. The most useful of
such Parameters include maximum rate of change of absorbance
(effectively the maximum slope observed in a plot of optical
density versus time) and time of onset of development (the time
after the beginning of development at which a given increment of
optical density is observed), both of which can be calculated for
each pixel in the scanned gel image through use of multiple scans
yielding optical density (or transmittance) as a function of time
during the development of the gel. Alternatively, sophisticated
curve-fitting algorithms can be used to devise functions of
absorbance as a function of time that yield, for each pixel, a
derived parameter well-correlated with known differences in
abundance.
[0152] Multiple scans of the same gel can also be used to compare
protein samples, provided that the proteins of each sample are
labeled prior to electrophoresis with a dye or other substituent
that can be detected separately from other such labels. Multiple
samples labeled with a series of different fluorescent dyes having
distinct emission wavelengths, for example, can be mixed and
co-electrophoresed. By using appropriate optical filters to detect
these dyes (and thus the proteins to which they are bound)
separately, the protein content of each sample can be measured
separately from the protein contents of other samples applied to
the same gel. When used in a 2-D procedure that includes
isoelectric focusing, such labels must be attached to the protein
in such a way that the protein's net pI is unaffected: if, for
example, the label is attached by reaction with a lysine primary
amino group, then the label must have a net charge of +1 to
compensate for the single positive charge of the primary amino
group lost when the lysine is derivatized. While this approach
increases the information output of each separation (by multplexing
samples), it also makes possible a substantial increase in net
resolution available for the comparison of samples. This comes
about because the different label distributions observed in a small
gel region (a protein spot in a 2-D electrophoresis pattern) can be
compared with great sensitivity by mathematical techniques to
determine whether the shape and location of a spot in one label
channel is precisely the same as the shape and location of a spot
in another label channel (both labels being detected on the same
gel where they reveal the proteins of two different samples). Spot
positional differences detectable by this approach (using for
example a correlation coefficient to determine whether the spot
profiles in two channels are the same or different) can be on the
order of 0.1 mm, far less than the 0.5-2.0 mm position difference
typically required to characterize protein spots as being different
when two different gels are compared, or when two samples are
co-electrophoresed on one gel and stained with a single stain. When
applied to both dimensions of a 2-D procedure, this method of
comparing potentially co-electrophoresing proteins can result in an
effective 100-fold increase in net gel resolution (the product of
an approximate 10-fold resolution increase in each dimension). Such
an approach is of particular value in comparing very different
protein patterns (for example different tissues), where it is
likely that different proteins with similar 2-D gel positions may
be encountered.
[0153] Spot Excision
[0154] Protein spots can be excised from the gel under computer
control once their positions are established by the aforementioned
scanning. FIG. 13 shows a mechanical cutter comprised of a block
156 in whose lower part a thin-wall tube 157 is mounted vertically
to act as a spot-cutting punch. The block and all its components
are mounted on a movable, computer controlled X-Y frame, suspended
just above and do-planar with the gel, such that the cutter 157 can
be positioned over any spot to be excised from the gel. A plunger
158 is arranged so as to move vertically within the punch. The
plunger extends through a hollow cavity 160 in the block and exits
through a second hole by means of channel containing an O-ring seal
159. The plunger is moved vertically by an actuator 161, and the
block is moved vertically by a second actuator 162 having less
force, and thus capable of being overridden by actuation of the
plunger actuator. The gel to be cut 97 lies horizontally on a flat
plat 163, which can be identical to the scanning platform/lightbox
146. In operation, the cutter performs a series of steps as shown
in the figure. In FIG. 13A, the block is positioned over the spot
to be cut. In FIG. 13B, the plunger actuator is pressed down,
forcing the plunger to protrude through the cutting tube 157 into
close proximity with the gel surface and further forcing the block
partially down through interaction of collar 164 on the plunger
with the block. In FIG. 13C, actuator 162 is forced down, forcing
the cutter through the gel and into contact with the supporting
plate 163. In FIG. 13D, the plunger actuator 161 is pulled upwards,
moving the block up by interaction of collar 164 with the block and
simultaneously generating suction in the cutter tube so as to
ensure that the cut gel plug 165 is lifted away from the gel by the
upwards motion. In FIG. 13E, the cutter has been repositioned over
a collection vessel 166, and the plunger forced down to expel the
gel plug into the vessel. In FIG. 13F, with both actuators in the
up position, a stream of wash liquid is introduced through hole 167
in the block 156 so as to expel any contaminating particulate gel
material remaining in the punch into a waste receptacle 168. Under
computer control, the spot cutting mechanism can excise hundreds of
spots from a single 2-D protein separation, depositing them in
96-well plates or other vessels for subsequent analysis by other
means such as mass spectrometry. In the preferred embodiment, the
spot cutter mechanism is incorporated into the gel scanning system,
thus allowing the gel to be cut in an automated fashion immediately
following computer analysis of the gel image obtained from the
scanner.
[0155] System Scheduling Algorithms
[0156] Operating as a continuous production line, the automated 2-D
gel system described must allow flexible scheduling of each
component action in the multi-step process required to make and run
each gel. If every gel were run using the same protocol, it would
be possible to design a completely synchronous scheduling system in
which each action recurred at precisely defined intervals. However,
such a system is inherently inflexible and would not allow running
successive gels with different parameters (e.g., different IPG pH
gradient, focusing volt-hours, or time in a stain solution). In
addition, any temporary halt required in such a synchronous system,
due for example to an equipment breakdown, would cause variable and
unforeseen consequences at different stages of the process.
[0157] Hence in the preferred embodiment, a non-synchronous
scheduling algorithm is used in which a series of steps is laid out
for the first sample to be run, and these are entered into a
database of actions required, each step associated with a relative
or absolute time at which it should be executed. Then a second
series of steps is laid out for the second sample to be run, and
these are entered into the database including a start delay
calculated so as to prevent any action required for the second gel
from being interfered with by any action required for the preceding
(first) gel. Additional gels are added in order by the same
procedure, ensuring in each case that the actions required for a
gel do not interfere with those required for previously entered
gels. Actions to be entered include casting an IPG gel,
transporting an IPG from the caster to a wash slot, transporting an
IPG from a wash to a drying slot, casting a slab gel, moving a slab
gel from mold to running slot, moving a slab gel from a running
slot to a stain slot, etc. Database entries take account of the
time required to execute such actions, e.g., the time to move a gel
from one station to another or to empty and refill a stain tank.
The sequence of operations required to effect the processing of a
series of gels, including interleaving of actions on different
gels, is readily obtained by retrieving from the database a series
of steps sorted by time of scheduled execution. Making use of the
ability of database software to sustain multiple independent
queries, different software modules controlling specific parts of
the hardware system may retrieve a subset of actions (in scheduled
time order) appropriate to them.
[0158] The automated system is then operated under the control of
one or more computer programs which function by examining the
database of scheduled actions, selecting from the database those
actions appropriate to the hardware components being controlled by
that program, and executing them at the time specified in the
appropriate database record. Hence, a single IPG manipulation arm
will be caused to transport IPG gels at different stages of the
process between the required slots and stations, actions on
different gels thus being interleaved in a flexible manner. Since
each gel is separately scheduled at the outset, it can have a
different protocol or different parameters than the preceding or
succeeding gel, without limitation.
[0159] Data Reduction
[0160] Scanned images of 2D protein patterns are subjected to an
automated image analysis procedure using a batch process computer
software (e.g., Kepler.RTM. software system). This software
subtracts image background, detects and quantitates spots, and
matches spot patterns to master 2D patterns to establish spot
identities. The final data for a 2-D gel, a series of records
describing position and abundance for each spot, are then inserted
as records in a computerized relational database.
[0161] Other Uses and Embodiments
[0162] The methods disclosed herein can be used for a series of
alternative analytical applications including the analysis of DNA
and RNA, as well as peptides. Either the automated IPG or slab gel
system can be used for high-throughput one-dimensional analyses of
relevant biomolecules as well as for 2-D.
[0163] It will be appreciated that the methods and structures of
the present invention can be incorporated in the form of a variety
of embodiments, only a few of which are described herein. It will
be apparent to the artisan that other embodiments exist that do not
depart from the spirit of the invention. Thus, the described
embodiments are illustrative and should not be construed as
restrictive.
LIST OF REFERENCES
[0164] 1. Laemmli, U.K. (1970) Nature 227, 680.
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Two-Dimensional Electrophoresis"), Feb. 13, 1997.
[0181] 18. Disclosure Document No. 393754 (Anderson, N. L.,
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[0183] 20. Disclosure Document No. 346229 (Anderson, N. L.,
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Apparatus"), Jan. 19, 1994.
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