U.S. patent application number 10/861787 was filed with the patent office on 2005-02-17 for method for treatment of a semisolid material.
This patent application is currently assigned to Invitrogen Corporation. Invention is credited to Amshey, Joseph W., Bogoev, Roumen A., Whitney, Scott E..
Application Number | 20050034989 10/861787 |
Document ID | / |
Family ID | 22816646 |
Filed Date | 2005-02-17 |
United States Patent
Application |
20050034989 |
Kind Code |
A1 |
Bogoev, Roumen A. ; et
al. |
February 17, 2005 |
Method for treatment of a semisolid material
Abstract
An apparatus and method for mincing a gel includes a gel mincing
tube and a mesh material. The mesh material extends across the end
of the tube. To subdivide a gel using the mincing apparatus, a gel
is placed upon the mesh material in the mincing tube, the mincing
tube, mesh material and the gel are spun in a centrifuge, forcing
the gel through the mesh material so that the gel is subdivided
into generally uniform smaller fragments. The mesh material may be
secured to a tube in the form of a nesting tube. The nesting tube
nests within the opening of a recovery vessel. The mesh material
may be placed in series with a conditionally porous membrane in the
nesting tube. Centrifuging the nesting tube and the recovery vessel
subdivides gel material into fragments by forcing the gel through
the mesh material. The gel subsequently falls upon the membrane,
and may be treated on the membrane to extract or otherwise treat
analytes in the gel material.
Inventors: |
Bogoev, Roumen A.; (Sam
Marcos, CA) ; Whitney, Scott E.; (San Diego, CA)
; Amshey, Joseph W.; (Encinitas, CA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Invitrogen Corporation
|
Family ID: |
22816646 |
Appl. No.: |
10/861787 |
Filed: |
June 4, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10861787 |
Jun 4, 2004 |
|
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|
09906792 |
Jul 18, 2001 |
|
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60218821 |
Jul 18, 2000 |
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Current U.S.
Class: |
204/450 ;
204/600 |
Current CPC
Class: |
Y10T 436/25375 20150115;
C07K 1/34 20130101; Y10T 436/2575 20150115; Y10T 436/255
20150115 |
Class at
Publication: |
204/450 ;
204/600 |
International
Class: |
G01L 001/20 |
Claims
1-10. (cancelled)
11. A method for the treatment of a semisolid material using a
first treating tube having a conditionally porous material disposed
therein, comprising the steps of: (a) combining the semisolid with
reactants in the first treating tube to create a reaction mixture;
and (b) centrifuging the first treating tube, such that some or all
of the reaction mixture is drawn through the conditionally porous
material.
12. The method of claim 11, further compromising: (c) placing the
first treating tube in a recovery vessel, such that the first
treating tube is nested into the recovery vessel; and (d) capturing
the reaction mixture in the recover vessel.
13. The method of claim 12, further comprising: (e) providing a
second treating tube for nesting with the recovery vessel, wherein
the second treating tube includes a second conditionally porous
material; (f) nesting the first treating tube in the second
treating tube and nesting the second treating tube in the recovery
vessel; (g) centrifuging the first and the second treating tubes;
and (h) capturing the reaction mixture in the recovery vessel.
14. The method of claim 11, wherein the conditionally porous
material comprises a ployvinylidene difluoride membrane or a nylon
membrane.
15. The method of claim 11, wherein the first treating tube
comprises an array of treating tubes for aligning and mating with a
microtiter plate.
16. The method of claim 11, wherein the first treating tube
includes a mesh material at a first end of a lumen of the first
treating tube, and the conditionally porous material is disposed at
a second end of the lumen of the first treating tube.
17. The method of claim 11, wherein the reactants are disposed on
the conditionally porous material, and wherein the reactants
comprise one or more components selected from the groups consisting
of a long-chain alkyl group, an ion exchange group, an antibody, a
short chain carboxylate or sulfonate, a chelating group and a
boronic acid.
18. The method of claim 11, wherein the semisolid material
comprises an electrophoresis gel, or a subportion thereof.
19-58. (cancelled).
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of the filing date of
U.S. Application No. 60/218,821, filed Jul. 18, 2000, the entire
disclosure of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Gel electrophoresis is a common procedure for the separation
of biological molecules, such as deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), polypeptides and proteins. In gel
electrophoresis, the molecules are separated into bands according
to the rate at which an imposed electric field causes them to
migrate through a filtering gel.
[0003] The basic apparatus used in this technique consists of a gel
often enclosed in a glass tube or sandwiched as a slab between
glass or plastic plates. The gel has an open molecular network
structure, defining pores, which are saturated with an electrically
conductive buffered solution of salts. These pores through the gel
are large enough to admit passage of the migrating
macromolecules.
[0004] The gel is placed in a chamber in contact with buffer
solutions which make electrical contact between the gel and the
cathode and anode of an electrical power supply. A sample
containing the macromolecules and a tracking dye is placed on top
of the gel. An electric potential is applied to the gel causing the
sample macromolecules and tracking dye to migrate toward the bottom
of the gel. The electrophoresis is halted just before the tracking
dye reaches the end of the gel. The locations of the bands of
separated macromolecules are then determined. By comparing the
distance moved by particular bands in comparison to the tracking
dye and macromolecules of known mobility, the mobility of other
macromolecules can be determined. The size of the macromolecule can
then be calculated or macromolecules of different sizes can be
separated in the gel.
[0005] There are a wide range of gel-forming materials used for
electrophoresis. Polyacrylamides, polymethacrylamides and other
related polymers are preferred for separation of smaller molecular
weight materials such as proteins, peptides and small nucleic
acids. Conversely, agarose, cellulose acetate and starch are
preferred for larger molecules. These gel materials are typically
compatible with aqueous systems, though some are also compatible
with non-aqueous solvents.
[0006] Formation of the gel material to the desired physical
dimensions can be accomplished by varying techniques, depending on
the material chosen. With agarose or gelatin the common method is
to heat the polymer causing the material to go into solution. The
solution can then be poured into a cast and allowed to polymerize
by cooling. Alternatively, polyacrylamides, polymethacrylamides and
other related polymers can be chemically polymerized by various
means including free radical induced polymerization with ammonium
persulfate and tetramethylethylenediamine.
[0007] A common problem with gel electrophoresis is the difficulty
of removing the protein, nucleic acid or other analyte of interest
from the gel after it has been separated from other components.
Because the gel matrix has very small pore sizes, large molecules
do not easily diffuse out of the gel matrix after they have been
drawn into the matrix through electromotive force. Proteins also do
not diffuse into the gel matrix readily in the absence of
electromotive force. The larger the molecular weight of the protein
the more difficult it is to get the molecules into or out of the
gel. Thus, techniques have been developed for preparing a gel for
extraction or introduction of molecules out of or into the gel.
[0008] The state of the art includes a procedure for gel
subdivision using a sieve. Christoph Eckerskorn and Rudolf Grimm
describe the use of a stainless steel sieve placed in the end of a
syringe barrel for subdividing gels. Eckerskorn and Grimm attribute
their technique to J. Heukeshoven and R. Demick, as described in B.
Radola (ed.), Electrophoresis Forum '91, Technical University,
Munich 1991, pp. 501-506. In another article, J. Lila
Castellanos-Serra, et al., Electrophoresis 1999; 20: 732-737, a
stainless steel sieve screen is used for subdividing gels to remove
proteins for further analysis. This article attributes the idea to
Eckerskorn and Grimm and to Heukeshoven and Demick.
Castellanos-Serra et al. placed a piece of stainless sieve screen
in the narrow end of a syringe barrel and used the pressure of the
plunger to force the gel through the screen.
[0009] However, this approach has significant disadvantages. First,
the syringe is costly, especially if there are large numbers of
gels to be processed simultaneously. This cost arises in part
because the syringe comprises several parts including a barrel,
plunger and gasket. Second, the user must manually force the gel
through the mesh with the action of a plunger. This technique is
significantly labor intensive and is not amenable to automation.
Third, the plunger does not advance all of the gel cleanly through
the mesh, because the force applied by the plunger stops at the top
of the mesh.
[0010] Others have demonstrated that centrifugal force can be
useful in forcing materials through a barrier for various purposes,
e.g. a filtration membrane for separation. For example, U.S. Pat.
No. 3,583,627 to Wilson describes concentrating a large molecular
weight substance in solution by fixing a filter into the end of the
upper of two nested tubes and spinning the tubes to force the
solvent through the filter while retaining the macromolecules.
There are numerous examples of using this basic principle to
concentrate macromolecules, for example U.S. Pat. No. 4,632,761
Bower et al. These devices and methods, however, are designed for
filtration of solutions and are not suitable for cutting or
subdividing a semi-solid gel or other substance.
[0011] Additionally, Millipore Corporation of Bedford, Mass.
currently sells Product No. 42600 "Ultrafree DA for DNA
extraction," for the subdivision of gel fragments. This product
also suffers from several drawbacks. First, it uses a nested tube
set, the upper tube having in its base plastic projections molded
in place that are supposed to subdivide a gel when the tube set is
spun. However, in practice, these do not work well because the
resulting subdivided gel has large, inconsistently-sized pieces of
gel leading to inefficient and unreliable extraction. Second, the
Millipore device is recommended primarily for agarose gels and may
be used on polyacrylamide gels, but only with a maximum polymer
concentration of 10% by weight. Gels having a polymer concentration
less than 10% by weight are usually unable to efficiently separate
very low molecular weight peptides and proteins, which often
require 12%, 15%, 18% or a higher percentage of polymer
concentration by weight.
[0012] There is, therefore, a need for a method of easily,
efficiently, reliably and inexpensively subdividing a gel to
facilitate the extraction of various molecules from the gel, or
conversely, the introduction of molecules into the gel. In
particular, as more and more analytes become available for study,
e.g. through the Human Genome Project and follow-on projects to
identify genes and express gene products, there is a need to
perform such extractions or introductions in high-throughput and
automated formats.
[0013] Although much of the description of the invention is related
to removing proteins from gels, it is also often of interest to
remove other molecules from gels. A person of ordinary skill in the
art would readily apply the techniques described herein to other
molecules commonly subjected to electrophoresis, e.g. nucleic acids
such as DNA or RNA.
SUMMARY OF THE INVENTION
[0014] The present invention provides a convenient method of
subdividing a gel containing a protein, nucleic acid or other
analyte of interest into small, consistently-sized fragments, which
facilitate the diffusion of reagents into, or analyte out of, the
gel. In one embodiment, the apparatus of the present invention
consists of a centrifuge tube incorporating a mesh or grid barrier,
through which the gel is drawn by centrifugal force when the tube
is spun, thereby forming a mincing tube. The mesh or grid is
preferably one having small and consistent hole spacing within and
between different manufacturing lots.
[0015] The mesh material extends across the end of the tube. To
subdivide a gel using the mincing tube, a gel is placed upon the
mesh material and the tube. When the mesh material and the gel are
spun in a centrifuge, the gel is drawn through the mesh material so
that the gel is subdivided into generally uniform smaller
fragments.
[0016] The mesh material may be secured to a tube in the form of a
nesting tube. The nesting tube nests within the opening of a
recovery vessel. Alternatively, the mesh material may be placed in
series with a conditionally porous membrane in the nesting tube.
Centrifuging the nesting tube and the recovery vessel subdivides
gel material into fragments by forcing the gel through the mesh
material. The gel subsequently falls upon the membrane, and may be
treated on the membrane to extract or otherwise treat analytes in
the gel material.
[0017] In an alternate embodiment, the centrifuge, tube may
comprise parts or segments, with one part of the nested set
including the mesh material to subdivide the gel. Another part
provides a porous membrane or a conditionally porous membrane.
Another part provides reversed phase capture material, either held
in place by a membrane or by using membrane derived so as to bind
proteins by hydrophobic interactions. Another part provides the
immobilized antibody to capture a high abundance protein. Several
types of such immobilized antibodies might be provided either as
separate segments for nesting or combined in a single segment. The
last segment can be a receptacle or recovery vessel for fluid
driven through the column by centrifugal force.
[0018] A particular advantage of this invention is that it makes
the process of subdividing the gel simple and suitable for
automation. It is often the case that numerous samples must be
processed for further analysis, such as determining their amino
acid sequence. The device and method of the present invention
avoids significant hands-on work, such as using a spatula to
chop-up or crush a gel, prior to the extraction process.
[0019] One advantage of the present invention is that it
significantly decreases the time required to elute a protein, or
other analyte, from the gel or to get homogeneous distribution of a
reagent being diffused into the gel. This advantage is achieved
because the invention results in a very finely subdivided gel.
Because the protein or other analyte must diffuse out of the gel
matrix, the farther it must diffuse, the longer it takes to
extract. Therefore, a very finely subdivided gel allows proteins,
for example, to be quickly diffused into or out of a gel.
[0020] Further, the present invention results in consistent
thickness of the gel fragments, which allows the diffusion distance
for reagents going into the gel or diffusing out of the gel to be
consistent. The time required for penetration of the fragments of
subdivided gel by reagent, or diffusion of materials out of the
fragments, will be consistent and reproducible only if the sizes of
the fragments are consistent and reproducible. Gels subdivided
manually will not have this consistency and therefore the amount of
protein, for example, which diffuses out of the gel will be
variable leading to inconsistent results in further
characterization. Often, samples of gel contain only tiny
quantities of protein to be used for subsequent characterization,
so consistency and efficiency of elution from the gel matrix is
very important.
[0021] Thus, the method and device of the present invention offer
several advantages. First, the syringe described by Eckerskorn et
al. is far more costly than a centrifuge tube. Second, centrifuging
methods, as described in the present invention, are more readily
and cheaply adaptable to automation than methods employing
syringe/plunger-and-mesh devices. Third, the use of centrifugal
force on the small gel fragments draws the fragments completely
through the sieve and down into the collecting tube, thereby
solving the problem in the prior art of gel material remaining in
the syringe mesh after the motive force of pressure is removed. In
contrast to the Millipore product (Product No. 46200) with its
molded projections for subdividing the gel, the sieve approach of
the present invention results in smaller and more consistent gel
fragments resulting in more efficient and reliable analyte
extraction. The present invention also, unlike the Millipore
product, subdivides gel with polyacrylamide concentrations greater
than 10%, because the centrifugal force effectively drives the gel
through the sieving mesh material.
[0022] In additional specific embodiments, methods and apparatuses
of the invention are used to subdivide gels having polyacrylamide
concentrations ranging from about 3% to 10%, about 5% to 10%, about
10% to 11%, about 10% to 12%, about 10% to 15%, about 10% to 18%,
about 10% to 20%, about 12% to 15%, about 12% to 18%, about 12% to
20%, about 15% to 18%, about 15% to 20% and about 18% to 20%.
Likewise, in other specific embodiments, the invention subdivides
any other gel types, including, but not limited to, agarose
gels.
[0023] In one aspect of the invention, a method for subdividing a
semisolid material using a mincing tube having a mesh material
disposed therein, comprises the steps of: placing the semisolid
material upon the mesh material of the mincing tube; and
centrifuging the mincing tube, the mesh, and the semisolid material
to facilitate passage of the semisolid material through the mesh,
thereby subdividing the semisolid material into fragments. Further
steps may comprise: introducing an extraction solution into the
mincing tube to extract an analyte from the semisolid material;
incubating the mincing tube including the extraction solution and
the semisolid material; and eluting the analyte from the subdivided
semisolid material, wherein the extracted analyte may be a
macromolecule, or alternatively, at least one or more of: proteins,
peptides, nucleic acids and carbohydrates. Furthermore, the mesh
material may span a lumen of the mincing tube, and the mesh
material may be concave from a top edge of the mincing tube.
[0024] Further steps may include introducing an extraction solution
into the mincing tube to extract analytes from the semisolid
material, wherein the extraction solution and the analytes create
an analyte solution; and transferring the analyte solution into a
recovery vessel, the recovery vessel having a conditionally porous
material disposed therein, such that the analyte solution may be in
contact with the conditionally porous material. Additionally, the
method may include centrifuging the recovery vessel with the
analyte solution, such that some or all of the analyte solution
flows through the conditionally porous material, wherein the
conditionally porous material comprises one or more components
selected from the group consisting of a long-chain alkyl group, an
ion exchange group, a short chain carboxylate or sulfonate, an
affinity group (e.g., an antibody), streptavidin, a chelating group
or a boronic acid. In one aspect of the method, the conditionally
porous material may be a polyvinylidene difluoride membrane a nylon
membrane, a nitrocellulose membrane and/or a glass fiber membrane
and the semisolid material may be an electrophoresis gel, or a
subportion thereof.
[0025] In another aspect of the invention, a method for the
treatment of a semisolid material using a first treating tube
having a conditionally porous material disposed therein, comprises
the steps of: combining the semisolid material with reactants in
the first treating tube to create a reaction mixture; and
centrifuging the first treating tube, such that some or all of the
reaction mixture may be drawn through the conditionally porous
material. The method may further comprise placing the first
treating tube in a recovery vessel, such that the first treating
tube may be nested into the recovery vessel; and capturing the
reaction mixture in the recovery vessel. Alternatively, the method
may further comprise: providing a second treating tube for nesting
with the recovery vessel, wherein the second treating tube includes
a second conditionally porous material; nesting the first treating
tube in the second treating tube and nesting the second treating
tube in the recovery vessel; centrifuging the first and the second
treating tubes; and capturing the reaction mixture in the recovery
vessel. The conditionally porous material may be a polyvinylidene
difluoride membrane, a nylon membrane, a nitrocellulose and/or a
glass fiber membrane and, in one aspect of the method, the first
treating tube may be an array of treating tubes for aligning and
mating with a microtiter plate. The first treating tube may include
a mesh material at a first end of a lumen of the first treating
tube, and the conditionally porous material may be disposed at a
second end of the lumen of the first treating tube. The reactants
may be disposed on the conditionally porous material, and the
reactants may comprise one or more components selected from the
groups consisting of a long-chain alkyl group, an ion exchange
group, a short chain carboxylate or sulfonate, an affinity group
(e.g., an antibody), streptavidin, a chelating group or a boronic
acid. Furthermore, the semisolid material may be an electrophoresis
gel, or a subportion thereof.
[0026] Another aspect of the invention includes a method for the
division of a semisolid material using a mincing tube having a mesh
material disposed therein, wherein the mincing tube may be nested
in a recovery vessel such that substances passing through the mesh
material are captured in the recovery vessel, the method comprising
the steps of: placing a semisolid material in the mincing tube; and
centrifuging the mincing tube and the recovery vessel until the
semisolid material is divided into fragments by the mesh material.
The method may further comprise the step of: providing a treating
tube nested in series after the mincing tube and before the
recovery vessel, wherein the treating tube includes a conditionally
porous material disposed therein, and the conditionally porous
material is in series with the mesh material. Further, the
conditionally porous material may comprise one or more of the group
consisting of: a long-chain alkyl group, an ion exchange group, a
short chain carboxylate or sulfonate, an affinity group (e.g., an
antibody), streptavidin, a chelating group or a boronic acid.
[0027] An apparatus for the subdivision of semisolid materials, may
comprise: a mincing tube; and a mesh disposed in the mincing tube,
wherein when the mincing tube is subjected to centrifugal forces, a
semisolid material placed within the mincing tube on one side of
the mesh is drawn through the mesh. The semisolid material may be
an electrophoresis gel, or a subportion thereof, and may contain a
protein or nucleic acid. Furthermore, the gel may have a
polyacrylamide concentration greater than 10 percent or less than
10 percent. The mesh of the apparatus may be a metal or a polymeric
mesh, such as a stainless steel mesh or a nylon mesh. The mesh may
have a hole size ranging from 0.01 mm.sup.2 to 9 mm.sup.2, and the
size of holes in the mesh may be substantially uniform. The mesh
may cover an end of the mincing tube, and may be flat or formed to
extend concavely into the mincing tube. The mesh may be fixed to
the mincing tube by welding, by an adhesive, or by a compression
ring.
[0028] The apparatus may be in a kit including a buffered solution
(which may or may not comprise one or more extraction reagents such
as one or more enzymes or the like), printed instructions for use
of the apparatus, a spare mesh material, particles treated with, or
having affixed thereto, an immobilized antibody, and a treating
tube containing a conditionally porous mesh material disposed
therein.
[0029] Another embodiment of the apparatus for the recovery of
proteins and nucleic acids from a gel comprises: a mincing tube
having a lumen, the mincing tube including a first conditionally
porous material extending across the lumen and a mesh material
extending across the lumen; and a recovery vessel disposed adjacent
to the mincing tube, such that contents of the mincing tube flow
through the mesh material and the conditionally porous material
into the recovery vessel. The mincing tube may be nested within the
recovery vessel. Additionally, the apparatus may include a treating
tube containing a, second conditionally porous material, wherein
the treating tube is disposed adjacent the mincing tube so that the
contents flow through the mesh material, the first conditionally
porous material and the second conditionally porous material in
series. The mincing tube may be nested within the treating tube,
and the treating tube may be nested within the recovery vessel. In
one aspect of the invention, the mincing tube and the treating tube
are arrays of tubes that align with and mate with a microtiter
plate. Contents of the apparatus may include an extraction buffer
and proteins. Further, the first conditionally porous material may
contain one or more of immobilized enzymes (e.g., proteases such as
trypsin, chymotrypsin, pepsin, papain and the like), immobilized
carbon chains and immobilized antibodies. The first conditionally
porous material may be a polyvinylidene difluoride membrane, a
nylon membrane, a nitrocellulose membrane and/or a glass fiber
membrane. In one aspect, the mincing tube may comprise: a first
portion containing the mesh; and a second portion containing the
first conditionally porous material, such that the contents of the
mincing tube flow through the mesh and the first conditionally
porous material in series. The first portion may be nested with the
second portion and the second portion may be nested with the
recovery vessel. The first and the second portions may be arrays of
tubes for aligning with and mating with a microtiter plate. The
mesh material may be a metal or fabric mesh, wherein the mesh is a
stainless steel mesh or a nylon mesh having a hole size ranging
from 0.01 mm.sup.2 to 9 mm.sup.2.
[0030] Another embodiment of an apparatus for subdividing and
processing a gel comprises: a mincing tube having a mesh material
disposed therein; and a recovery vessel connected to the mincing
tube, wherein a conditionally porous material is disposed within
the recovery vessel. A reagent may be attached to the conditionally
porous material, and the reagent may be one of immobilized trypsin,
immobilized carbon chains and immobilized antibodies. The recovery
vessel may be removably connected with the mincing tube in an
inverted relationship. The mesh may a metal or fabric mesh, such as
a stainless steel mesh or a nylon mesh having a hole size ranging
from 0.01 mm.sup.2 to 9 mm.sup.2. The conditionally porous material
may be a polyvinylidene difluoride membrane, a nylon membrane, a
nitrocellulose membrane and/or a glass fiber membrane.
BRIEF DESCRIPTION OF THE FIGURES
[0031] The foregoing and other features and advantages of the
invention will be apparent from the following, more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings.
[0032] FIG. 1 shows a gel mincing tube including a mesh
material.
[0033] FIG. 2 shows a nested gel mincing tube including a mesh
material.
[0034] FIGS. 3A-3D show the use of the nested tube of FIG. 2,
including processing of the sub-divided gel and a basic recovery
technique.
[0035] FIGS. 4A-4D show the use of an alternative embodiment of a
gel mincing tube, including an alternative method of recovery by
inversion of the gel mincing tube into a recovery vessel.
[0036] FIG. 5 shows an alternative embodiment of a nested gel
mincing tube, including a mesh material bottom and a treating
membrane.
[0037] FIG. 6 shows an alternative embodiment of a nested gel
mincing tube with a nested treating tube and recovery vessel.
DETAILED DESCRIPTION OF THE INVENTION
[0038] Preferred embodiments of the present invention are now
described with reference to the figures where like reference
numbers indicate identical or functionally similar elements. A
person skilled in the relevant art will recognize that other
configurations and arrangements can be used without departing from
the spirit and scope of the invention. Furthermore, although the
invention is described with reference to subdividing and treating
electrophoresis gels, or subportions thereof, the invention is
further applicable to any semisolid material, with the only
limitation on the material being that it is subdividable through a
mesh or screen material. Additionally, although the description of
the invention primarily describes the invention as recovering
proteins from the gels, the present invention can be used to
recover other substances, including, but not limited to, nucleic
acids, peptides, oligonucleotides and carbohydrates, and
combinations thereof (e.g., glycoprotein, proteoglycans, nucleic
acids, peptide-nucleic acid complexes and the like). Alternatively,
the invention can be used to recover any macromolecule.
[0039] As used herein, the term "macromolecule" refers to polymeric
and non-polymeric molecules which are larger than a specified size
(see below) or contain more than a specified number of monomeric
units (see below). Non-polymeric macromolecules will typically have
a molecular weight over about 10,000 daltons. Further, polymeric
macromolecules, such as proteins, will typically comprise more than
25 amino acids. Polymeric macromolecules, such as nucleic acids,
will typically comprise more than 40 nucleotides. Polymeric
macromolecules, such as multimeric carbohydrates, will typically
comprise more than 10 individual sugar monomers. Polymeric
macromolecules, other than those described above, would be apparent
to one skilled in the art and may include combinations of the
polymeric molecules described above (e.g., peptide-nucleic acids).
The term "macromolecule" does not include molecules of the
particular gel (e.g., agarose, polyacrylamide, etc.) from which
analytes are extracted.
[0040] As used herein, the term "peptide" refers to polymers
composed of 25 or fewer amino acids.
[0041] As used herein, the term "oligonucleotide" refers to
polymers composed of 40 or fewer nucleotides.
[0042] As used herein, the term "purified," when applied to a
particular molecule, means that the molecule is separated from at
least some surrounding contaminants. Contaminants include molecules
which are normally associated with the molecule which is purified.
For example, an intracellular protein is purified when it is
separated from at least some nucleic acid and protein molecules
which normally co-reside with it in a cell. As one skilled in the
art would recognize, the term "purified" is relative and, in
essence, means that the concentration of a molecule, with respect
to other molecules with which it is normally associated, is
increased. For example, passage of a protein mixture over a
molecular weight column may result in dilution of a particular
protein (i.e., the protein which is being purified) and an increase
in the concentration of this protein with respect to other protein
present in the original mixture. Thus, generally, the term purified
does not mean that the molecule subjected to processes which lead
to purification is separated from reagents such as buffers or
compounds such as water.
[0043] As used herein, the term "isolated," when applied to a
particular molecule, means that the molecule is separated from
substantially all of the surrounding contaminants. Thus, the term
"isolated" means that molecule being isolated is at least 95% pure,
with respect to the amount of contaminants originally present. In
other words, the molecule which is isolated is separated from at
least 95% of the surrounding contaminants.
[0044] The present invention uses a sieving screen to subdivide the
gel in an inexpensive format that lends itself to automation.
Screens of metal mesh material or fabric mesh material can be used
to subdivide the gel into uniformly small fragments. The screens
may have openings that range from 0.01 mm.sup.2 to 9 mm.sup.2.
Preferably, the openings each have an area from 0.09 mm.sup.2 to
0.36 mm.sup.2. The openings may be any shape, including square,
rectangular, oval, circular, triangular or any variation thereof.
Regardless of the shape of the openings, the area of the openings,
in specific embodiments ranges from about 0.01 mm.sup.2 to mm.sup.2
to 0.1 mm.sup.2, about 0.01 mm.sup.2 to 1 Mm.sup.2, about 0.01
mm.sup.2 to 2 mm.sup.2, about 0.01 mm.sup.2 to 9 mm.sup.2, about
0.01 mm.sup.2 to 5 mm.sup.2, about 0.1 mm.sup.2 to 1 mm.sup.2,
about 0.1 mm.sup.2 to 5 mm.sup.2, about 0.1 mm.sup.2 to 9 mm.sup.2,
about 1 mm.sup.2 to 5 mm.sup.2, about 1 mm.sup.2 to 9 m.sup.2 and
about 5 mm.sup.2 to 9 mm.sup.2.
[0045] Screens such as metal sieve, used to separate particles by
their size, and fabrics such as nylon mesh are commercially
available with openings from less than 0.09 mm.sup.2 to 0.36
mm.sup.2 or larger, and are highly consistent across multiple lots
of product. These screens and fabrics are available from numerous
vendors, including, but not limited to, McMaster-Carr, Santa Fe
Springs, Calif. 90670-2932. Such materials may be largely inert
when made of Appropriate materials such as stainless steel or
nylon. When the gel is mechanically forced through these materials,
it breaks into very small fragments with consistent thickness, (J.
Lila Castellanos-Serra, et al., Electrophoresis 1999; 20:
732-737).
[0046] The device can be used to capture intact protein by elution
from the gel fragments produced by centrifugation through the mesh
material. Conventionally, proteins are removed from gels by
electroelution, a method of passing a current through the gels to
remove them by the same mechanism which caused them to separate in
the gels originally. See Hunkapiller, M. W., et al., in Methods in
Enzymology, C. H. W. Hirs and S. N. Timasheff eds., Academic Press,
New York, Vol. 91, p. 227. However, when the gel is subdivided into
very small elements, a significant portion of the protein can be
eluted directly. Protein eluted in this way can be sequenced using
automated sequencers such as are available from Beckman Instruments
or Applera, Inc. among others.
[0047] FIG. 1 shows an embodiment of gel mincing apparatus 100 for
dividing a gel into subdivided fragments. Apparatus 100 includes a
gel mincing tube 102 and a screen or mesh material 104. Mincing
tube 102 can be a plastic centrifuge tube, as is shown in FIG. 1.
Mincing tube 102 has an open end 108 and a closed end 110, forming
an interior cavity 114. Mincing tube 102 could be a bowl, a dish, a
bucket or any vessel having an open end and a closed end, thereby
being capable of containing a fluid. As is explained with respect
to alternate embodiments below, the mincing tube may include two
open ends, with a lumen extending therebetween. In the embodiment
shown, mincing tube 102 is similar in shape to an Eppendorff
centrifuge tube, such that closed end 110 is in a cone shape.
[0048] Mesh material 104 extends across open end 108 of mincing
tube 102. Mesh material 104 may be perpendicular to a longitudinal
axis of mincing tube 102, or may have a concave shape extending
from a tube edge 112 into cavity 114, as is shown in FIG. 1. Mesh
material 104 may removably rest upon tube edge 112, or,
alternatively, may be fixed to tube edge 112. Mesh material 104
should be secured to mincing tube 102 in such a way that during
centrifuging, the forces imposed on mesh material 104 do not
advance the mesh further into cavity 114 toward closed end 110.
[0049] To subdivide a gel using mincing apparatus 100, a gel 106 is
placed upon mesh material 104 above cavity 114. Gel 106 could be
any gel, but preferably is a electrophoresis gel or a subportion
thereof. Mincing tube 102, mesh material 104 and gel 106 are spun
in a centrifuge, forcing gel 106 consistently through mesh material
104 so that the gel is subdivided into smaller fragments. Mesh
material 104 can be secured into open end 108 of mincing tube 102,
so that when gel 106 is placed on mesh material 104, the tube can
be placed into the centrifuge without spilling the gel.
[0050] Mesh material 104 can be inserted into mincing tube 102 so
that it is held in place by mechanical forces. Specifically, a
piece of stainless steel mesh material can be cut to be about twice
the diameter of the top of the mincing tube. The portion of the
mesh above the opening of the mincing tube is pressed into the
mincing tube so that the mesh is somewhat concave, and extends into
the opening. The edges of mesh 104 that extend outside of the mouth
of mincing tube 102 can be bent over to hold the mesh in place. Gel
sample 106 to be subdivided is placed in the concavity above
mincing tube cavity 114 and the entire assembly is centrifuged.
Alternative methods for holding the mesh in place include, but are
not limited to, gluing, welding or otherwise fixing the mesh to the
top of the mincing tube so that it hangs into the mincing tube
cavity. Fixing the mesh to the top of the mincing tube is useful
when using a flexible mesh, such as a nylon mesh. The mincing tube
may be made from a centrifuge tube available from Millipore
Corporation (Bedford, Mass.), Brinkmann (Westbury, N.Y.), Fisher
Scientific (Pittsburgh, Pa.) and others, as described below.
[0051] In lieu of mincing tube 108, a microtiter plate could be
used, having a mesh material disposed therein. Such use of a
microtiter plate allows high throughput and efficient subdividing
of a plurality of gel pieces.
[0052] FIG. 2 shows an alternate embodiment of a gel mincing
apparatus 200. In this embodiment, a mesh material 204 is secured
to a base of a nesting mincing tube 208. Mesh material 204 could be
secured to nesting mincing tube 208 by an adhesive, by a weld, tied
around nesting mincing tube 208, by a compression ring or attached
by any other means known in the relevant art. Alternatively,
mincing tube 208 could have a step formed into the tube lumen, and
mesh material 204 could be lodged across the lumen, on the
step.
[0053] Nesting mincing tube 208 has an outer diameter smaller than
the inner diameter of a recovery vessel 202, and is removably
placed within a cavity 214 of recovery vessel 202. In a preferred
embodiment, recovery vessel 202 is a centrifuge tube similar to
mincing tube 102 described above with reference to FIG. 1. A lip
216 is located around an upper end of nesting mincing tube 208, and
radially extends from the upper end of nesting mincing tube 208 to
a diameter greater than the inner diameter of recovery vessel 202.
Accordingly, lip 216 secures nesting mincing tube 208 into recovery
vessel 202, limits the distance that nesting mincing tube 208 can
extend into cavity 214 during centrifuging, and serves as a
convenient gripping point when removing nesting mincing tube 208
from recovery vessel 202. Nesting mincing tube 208 and recovery
vessel 202 are preferably formed of an inert plastic material.
However, as would be apparent to one skilled in the relevant art,
nested tube 208 and recovery vessel 202 could be formed of any
plastic, metal or other material.
[0054] Nesting mincing tube 208 is conveniently nested within
recovery vessel 202 during use. Likewise, when desired, a user
could remove nesting mincing tube 208 from recovery vessel 202 to
further aid in processing of a gel sample, as will be explained
below. Although nesting mincing tube 208 is shown having mesh
material 204 located at an end of the nesting tube, mesh material
204 could be secured within nesting mincing tube 208 in a middle
region or at the opposite end of that shown in FIG. 2. Preferably,
mesh material 204 is located at the lower end, as shown, or
somewhere in the middle region so that a gel 206 can be easily
placed upon mesh material 204 without spilling.
[0055] Nested tubes, as disclosed in FIG. 2, could be created by
modifying a standard centrifuge nesting tube set used for
concentration or ultrafiltration made by, for example, Millipore
Corporation and others. One method of creating the nested tube set
is to cut off a bottom of a standard, nesting tube in the region of
the ultrafiltration membrane, and to attach a mesh material by
gluing or by melting the nesting tube plastic so as to weld the
mesh material to the nesting tube. The nesting tube may then be
placed into the centrifuge tube of the standard nesting tube set,
the gel sample may be placed atop the mesh in the nesting tube and
the nested pair may be spun in a centrifuge. The subdivided gel
fragments then collect in the centrifuge tube where the fragments
may be subjected to further processing.
[0056] FIGS. 3A-3D show a process for extracting samples from a gel
using the gel mincing apparatus of FIG. 2. FIG. 3A shows the gel
mincing apparatus 200 of FIG. 2, including nesting mincing tube
208, mesh material 204 and recovery vessel 202. In use, a gel 206
is placed within nesting mincing tube 208 of mincing apparatus 200.
Apparatus 200 is centrifuged until gel 206 is drawn by centrifugal
force through mesh material 204 into cavity 214. As gel 206 is
drawn through mesh material 204, gel 206 is subdivided into a
plurality of substantially similarly sized subdivided fragments
310, as shown in FIG. 3B.
[0057] Following the subdivision of the gel by centrifugation
through the mesh, an extraction buffer solution 312 may be
introduced into recovery vessel 202, as is shown in FIG. 3C.
Although the invention is described with use of an extraction
buffer solution, the solution need not be a buffer solution, but
could be any extraction solution. Accordingly, extraction buffer
solution 312 may or may not include a buffer. Extraction buffer
solution 312 may be poured through the lumen of nesting mincing
tube 208 and through mesh material 204 into recovery vessel 202.
Commonly, extraction buffer solutions are prepared with volatile
salts, such as ammonium bicarbonate, which facilitates subsequent
freeze drying. However, any standard extraction solvent may be
used, as would be apparent to one skilled in the relevant art.
After introducing extraction buffer solution 312, the subdivided
gel sample may be incubated. The technical specifications for
incubation times and temperatures are known or determined
experimentally, as elution efficiency is dependent on the molecular
weight of the analyte, the amount of polymer per unit weight of gel
and the size of the gel fragments.
[0058] During incubation, extraction buffer 312 elutes analytes,
such as proteins, from gel subdivided fragments 310, creating a
buffer-analyte solution 314 as shown in FIG. 3D. Buffer-analyte
solution 314 may also include any analyte that is commonly
subjected to electrophoresis, including, for instance, proteins and
nucleic acids, such as DNA and RNA. After incubation, nesting
mincing tube 208 may be removed from recovery vessel 202, and a
pipette 316 may be used to obtain the eluted sample. Although
nesting mincing tube 208 was removed from recovery vessel 202 after
incubation, it would be equally obvious to remove nesting mincing
tube 208 prior to incubation or prior to introducing extraction
buffer solution 312 to recovery vessel 202.
[0059] Proteins eluted from the gel fragments may be used for
further analysis. For example, proteins are frequently examined for
their tendency to bind to other proteins indicating possible
interactions that may take place between these proteins within the
cell. Methods for evaluating such interactions include equilibrium
dialysis, surface plasmon resonance changes, and others. Moreover,
such eluted proteins may be used to evaluate interactions with
small molecules such as intracellular messenger compounds or
potential drug substances. If an eluted protein is important in the
regulation of cellular function, it is frequently desirable to
determine whether its function can be modulated with small
molecules that are potential drug substances.
[0060] FIGS. 4A-4D show an alternative embodiment of a gel mincing
apparatus 400, including an alternative method of recovery by
inverting the mincing tube into a recovery vessel. A gel mincing
tube 402 includes an open end 407 and a closed end 408. Similar to
the embodiments of FIGS. 1 and 2, a mesh material 404 is disposed
within mincing tube 402, spanning the diameter of mincing tube 402.
In this embodiment, mesh material 404 is shown spanning the
diameter in a central region of mincing tube 402. As would be
apparent to one skilled in the relevant art, mesh material 404
could be located at or near open end 407 or near closed end 408 of
mincing tube 402.
[0061] A gel 406 is placed within mincing tube 402 on mesh material
404, as is shown in FIG. 4A Mincing tube 402, mesh material 404 and
gel 406 are centrifuged until gel 406 passes through mesh material
404, and is thereby subdivided into gel fragments 410 as is shown
in FIG. 4B. An extraction solution 412 is introduced into mincing
tube 402, as is shown in FIG. 4B. As would be apparent to one
skilled in the relevant art, extraction solution 412 could be a
buffer solution. Mincing tube 402 is incubated with extraction
solution 412 and gel fragments 410 to draw analytes out of the gel
fragments, thereby creating an analyte solution 420. After
incubation, analyte solution 420 is poured from recovery vessel 202
into a recovery vessel 414. As can be seen in FIG. 4C, the
remaining gel fragments are restrained within mincing tube 402 by
mesh material 404.
[0062] Recovery vessel 414 may optionally include a membrane 416 of
polyvinylidene difluoride, nylon, nitrocellulose, glass fiber or
other porous material, disposed within a cavity 422. Recovery
vessel 414 preferably has an inner diameter greater than the outer
diameter of mincing tube 402 so that mincing tube 402 may be
inverted and placed within recovery vessel 414 to reduce spilling
and to allow the analyte solution to completely drip into recovery
vessel 414, as is shown in FIG. 4C. However, as would be apparent
to one skilled in the relevant art, the diameters of mincing tube
402 and recovery tube 414 could be the same, or the diameter of
mincing tube 402 could be smaller or larger than the diameter of
recovery vessel 414. Membrane 416 spans the diameter of cavity
422.
[0063] Mincing tube 402 may attach to recovery vessel 414 using any
standard technique. For example, mincing tube 402 may have a
peripheral lip (not shown), extending from a circumference of
mincing tube 402 from an area located between open end 407 and
closed end 408. Thus, the lip allows mincing tube 402 to rest
inverted on recovery vessel 414. Likewise, mincing tube 402 could
be friction fit into the inner diameter of recovery vessel 414,
thereby securing mincing tube 402 in an inverted position. In an
alternative embodiment, mincing tube 402 could be taped, clipped or
include threads that allow mincing tube 402 to be secured or
threaded onto recovery vessel 414.
[0064] When poured into recovery vessel 414, analyte solution 420
occupies the area above membrane 416. Membrane 416 is a
conditionally porous material, meaning that the pores of the
membrane are small enough to restrict passage of a solution through
the membrane except when a force is applied to the solution.
Accordingly, as is seen in FIG. 4C, analyte solution 420 is
contained in the area above membrane 416.
[0065] To filter and recover the analytes from analyte solution
420, recovery vessel 414 and mincing tube 402 are centrifuged.
During centrifuging, analyte solution 420 is drawn through the
pores of membrane 416 into cavity 422. Gel fragments 410 are again
drawn through mesh material 404, and fall onto membrane 416 as
shown in FIG. 4D. The centrifugation allows for complete recovery
of the eluted product and, simultaneously, membrane 416 separates
gel fragments 410 from the eluted product making the recovery more
pure.
[0066] In one embodiment, membrane 416 is a Biodyne C membrane from
Pall Gelman Corp. of Ann Arbor, Mich. A Biodyne C membrane is a
nylon 6,6 membrane with pore surfaces populated by a high density
of carboxyl groups. The Biodyne C membrane may be modified to
create a reverse phase surface. One way of modifying the membrane
is to incubate a 7 cm.times.7 cm membrane in 50 mL of 100 mM MES
(morpholinoethane sulfonic acid) pH 5.5 containing 20 mM EDC
(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) for
15 minutes at room temperature in a polypropylene container on a
rotary shaker. After incubating, 5 mL of 0.5 M N-hydrosuccinimide
in water (or sulfo-N-hydroxysuccinimide) is added and mixed
thoroughly for 20 minutes. The solution in decanted and the
membrane is washed with fresh MES buffer. The membrane is then
incubated with 50 mM hexadecyl amine in 0.1 M NaHCO.sub.3 pH 8.1
containing 50% tetrahydrofuran. The membrane is incubated for 2
hours at room temperature with shaking, and then the membrane is
washed for 3 successive washes with 0.1 NaHCO.sub.3 for 10 minutes,
followed by three 10 minute water washes. The membrane is allowed
to air dry. The Biodyne C membrane may be used by itself as a
cation exchange resin.
[0067] The Biodyne B membrane, also manufactured and sold by Pall
Gelman Corp., can be used as a strong anion exhange membrane for
capture of peptides or proteins. The Biodyne B membrane is a good
capture membrane for oligo nucleotides.
[0068] As would be apparent to one skilled in the relevant art,
mincing tube 402 need not be centrifuged while inverted above
recovery vessel 414. However, such joint centrifuging is desirable
because it aids in transferring all of the analyte solution from
mincing tube 402 to recovery vessel 414. Thus, this method allows
treating and processing without requiring that the mesh material be
removed.
[0069] Protein eluted from gel fragments 410 maybe captured by
membrane 416. Membrane 416 could be a polyvinylidene difluoride
membrane such as the Invitrolon P, available from Invitrogen Corp.,
Carlsbad, Calif., or other materials having affinity for protein
including those materials described elsewhere herein.
[0070] In an alternate embodiment, the membrane is placed in a
nested tube set such that the protein is spun through the membrane
to effect capture, as will be described below with reference to
FIG. 5. Captured proteins may be sequenced using the Edman method
on automated sequencers. See K. J. Wilson and P. M. Yuan, "Peptide
and Protein Purification", in Protein Sequencing: A Practical
Approach, J. B. C. Findlay and M. J. Geisow, eds., IRL Press,
Oxford, 1989, p. 1. Protein eluted from the subdivided gel
fragments is captured on the membrane and the membrane can be cut
out and placed directly in the automated sequencer device.
[0071] In other embodiments of the present invention, additional
features can be designed into the centrifugal gel mincing apparatus
to accomplish other preparation steps on the extracted analyte. In
particular, additional sample processing steps can be conducted in
the same or in nested sets of tubes. Such processing is
particularly desirable because once the gel fragments are
subdivided, they more readily lend themselves to treatments with
solvents and other reagents. This is due to the fact that the
smaller gel fragments allow the molecules (analytes) embedded in
them to more easily and quickly contact or diffuse into such
reagents.
[0072] For example, proteins isolated in gel slices are commonly
treated with trypsin or other oft-known proteolytic enzymes
(collectively referred to as proteases) to cleave them into
peptides for further analysis. See T. Rabilloud, et al.,
Electrophoresis 1999; 20, 3603-3610. Segments of the gel containing
protein, visualized as spots by use of protein stains, are excised,
chopped and dehydrated by air-drying or by using water miscible
volatile solvents such as acetonitrile. They are then rehydrated in
buffer containing trypsin or other proteases and digested for 15
hours at 37.degree. C. The peptides formed by the digestion of the
proteins are then extracted with mixed solvents.
[0073] The methods of the invention could be performed by proteases
digestion times of greater or less than 15 hours (e.g., about 30
minutes, about 1 hour, about 2 hours, about 5 hours, about 10
hours, about 20 hours, etc.), such as a range of time from about 7
hours to 20 hours, 9 hours to 20 hours, 12 hours to 20 hours, 14
hours to 20 hours, 16 hours to 20 hours, 9 hours to 16 hours, 12
hours to 16 hours, 14 hours to 16 hours, 9 hours to 14 hours, 12
hours to 14 hours and 9 hours to 12 hours. Likewise, proteases
digestion temperatures could range from about 25.degree. C. to
45.degree. C. (e.g., about 25.degree. C., about 30.degree. C.,
about 35.degree. C., about 37.degree. C., about 40.degree. C. deg,
etc.), about 30.degree. C. to 45.degree. C., about 32.degree. C. to
43.degree. C., about 32.degree. C. to 40.degree. C., about
32.degree. C. to 37.degree. C., about 32.degree. C. to 34.degree.
C., about 34.degree. C. to 42.degree. C., about 34.degree. C. to
37.degree. C. and about 37.degree. C. to 40.degree. C.
[0074] Trypsin, as well as other proteins, can be immobilized onto
a membrane, polymer or solid support such as glass particles using
a variety of common techniques. W. V. Bienvenut, et al., Analytical
Chemistry 1999; 71(21), 4800-4807 describes the immobilization of
trypsin onto a chemically modified polyvinylidene difluoride
membrane containing activated carboxylic acid groups. The protein
amino groups react with the activated carboxylic acids linking the
protein to the membrane. Many other immobilization techniques are
also well known in the art and are well described in Chemistry of
Protein Conjugation and Cross-Linking, by Shan S. Wong, CRC Press,
New York, 1993. The immobilized trypsin, or any other protease, can
then be placed in a layer beneath the subdivided gel, such that the
proteins eluted from the gel will interact with the layer thus
being cleaved into peptides without contaminating the sample itself
with free trypsin.
[0075] FIG. 5 shows an embodiment of a mincing tube apparatus 500
including a nesting tube 508 containing a mesh material 504 and a
treating membrane 516. Nesting tube 508 nests within a recovery
vessel 502 in the same nesting relationship described previously
with reference to FIG. 2. In this embodiment, a mesh material 504
is used to subdivide a gel, such as gel 506, into fragments.
Membrane 516 is located in series after mesh material 504. Membrane
516 could be a conditionally porous membrane, which, as stated
above, is a membrane having pores of small enough to restrict
passage of a solution through the membrane except when a force is
applied to the solution. Membrane 516 is also securely attached to
nesting tube 508. Mesh material 504 is located in a first portion
of nesting tube 508, and membrane 516 is located, spaced apart from
mesh material 504, within a second portion of nesting tube 508. The
space between mesh material 504 and membrane 516 allows gel 506 to
be drawn through mesh material 504 during centrifuging.
[0076] During the centrifuging process, gel 506 is divided by and
passes through mesh material 504 and accumulates on membrane 516.
In one embodiment, membrane 516 contains an immobilized protease,
such as trypsin. As the subdivided gel fragments accumulate on
membrane 516, the protease will begin to digest proteins that elute
from the gel fragments.
[0077] FIG. 6 shows an alternative embodiment of a gel mincing
apparatus 600. In this embodiment, gel mincing apparatus 600
includes a mincing tube 608 and a treating tube 612. Mincing tube
608 and treating tube 612 are nesting tubes, and as shown, mincing
tube 608 is nested within treating tube 612. A mesh material 604 is
secured in mincing tube 608, for subdividing a gel. As described
with respect to previous embodiments, mesh material 604 could be
located at the top, central area or bottom of mincing tube 608.
[0078] A conditionally porous membrane 616 is secured in treating
tube 612, for treating a subdivided gel or molecules contained
within the gel. Thus, when nested, mesh material 604 and membrane
616 are in series. Membrane 616 could be a conditionally porous
membrane, and/or may be modified by the attachment of long-chain
alkyl groups, ion exchange groups such as tertiary or quaternary
amines, short chain carboxylates or sulfonates, chelating groups
for metal ion affinity capture, boronic acids for capture of
carbohydrates, and similar modifications. Alternatively, membrane
616 may contain or support particles, such as ion exchange or
reversed phase capture particles on the membrane.
[0079] In the embodiment shown, treating tube 612 has a greater
longitudinal length than mincing tube 608. Accordingly, when nested
together, a gap 610 is formed between mesh material 604 and
membrane 616. Gap 610 preferably is large enough to fully contain a
fragmented gel, after a gel is minced by mesh material 604.
However, as would be apparent to one skilled in the relevant art,
treating tube 612 need not have a greater longitudinal length than
treating tube 612. In one embodiment, either mincing tube 608 or
treating tube 612 could have steps or ledges formed into the sides,
which would serve to limit the distance that mincing tube 608 could
be inserted into treating tube 612.
[0080] In another embodiment, the profiles of both mincing tube 608
and treating tube 612 are tapered. Thus mincing tube 608 easily
nests within treating tube 612. During manufacturing, gap size 610
may be determined by varying the angle of the taper and the size of
any ridges, ledges or steps that may be formed into the profiles of
mincing tube 608 and treating tube 612.
[0081] In another embodiment, a spacer (not shown) could be placed
between mincing tube 608 and treating tube 612, either around the
exterior or the interior of the tubes, to determine the size of gap
610.
[0082] Treating tube 612 is nested within a recovery vessel 602.
Recovery vessel 602 could be a centrifugal tube, as described with
respect to the previous embodiments. A cavity 614 is formed between
membrane 616 of treating tube 612 and an interior surface of
recovery vessel 602. Because mincing tube 608 and treating tube 612
are removably nested within each other, and removably nested within
recovery vessel 602, one nested tube could be used without the
other, or both nested tubes could be used together in series.
[0083] In use, a gel 606 is placed on mesh material 604. During
centrifuging, gel 606 is fragmented into smaller pieces, which
enter gap 610. If membrane 616 is a treating membrane, having
long-chain alkyl groups, ion exchange groups such as tertiary or
quaternary amines, affinity capture, agents (such as antibodies,
ligands, substances or other agents that bind proteins), short
chain carboxylates or sulfonates, chelating groups for metal ion
affinity capture, boronic acids for capture of carbohydrates, or
similar modifications thereon, treating of the gel, and the
substances in the gel occurs. If membrane 616 is a non-treating
membrane, an extraction solution, such as an extraction buffer, is
poured into treating tube 612. After treating, centrifuging draws
the extraction solution and the elected substances through membrane
616 into cavity 614 of recovery vessel 602.
[0084] In one alternate embodiment, one or more additional treating
tube are used in series with mincing tube 608 and treating tube
612. Accordingly, a user can use desired treatments on separate
membranes to perform a number of treatments in series, without
disassembling the apparatus.
[0085] In another alternate embodiment, multiple treating tubes are
used in series without gel mincer tube 604.
EXAMPLE 1
[0086] In one embodiment of the present invention, the trypsin is
immobilized onto microscopic glass particles containing carboxylic
acid groups like those available from Sigma Chemical Company, St.
Louis, Mo., Catalog Number G3910, p. 482 of the 1999 catalog. Glass
particles are activated using carbodiimide, See S. S. Wong, above,
and will bind proteins such as trypsin through the protein's amino
groups. These resulting particles of immobilized trypsin are placed
on a filter membrane or other similar support that will retain the
particles but allow the passage of solution and protein. The
immobilized trypsin membrane is placed into the lumen of the tube
such that it is below the mesh layer. These layers are placed in
series either by mechanically pushing the membrane into the tube
such that it is lower than the nesting mechanism, or by fixing the
trypsin layer to the nesting tube as described above, with
reference to FIG. 5.
[0087] When the mesh and the membrane containing the immobilized
trypsin particles are placed in series and spun, the gel is
subdivided through the mesh and the small bits of gel and solution
fall onto the filter holding the immobilized trypsin. This filter
may be a conditionally porous membrane such as a molecular weight
cutoff membrane sold by Pall Gelman Corp., of Ann Arbor Mich.
Proteins eluted from the gel are digested by the trypsin. The
resulting peptides and/or proteins are separated from the
immobilized trypsin particles and moved through the membrane into a
recovery vessel or capture tube by centrifugal force. The amount of
centrifugal force required to draw the peptides and proteins
through the membrane depends on the permeability, or molecular
weight cutoff, of the membrane supporting the immobilized trypsin.
If preferred, these multiple reactions of mincing and treating can
be run separately by using single function nested tube sets, such
as a mincing tube set and a treating tube set.
EXAMPLE 2
[0088] In another embodiment, the trypsin is immobilized onto the
membrane itself, such as with the carboxylate modified
polyvinylidene difluoride membrane described by Bienvenut et al.
The treated membrane can be glued or welded into the bottom of the
second nesting centrifuge tube and used in the same way as noted
above.
[0089] One advantage to using immobilized trypsin, is that by
immobilizing the trypsin, the enzyme will not digest other trypsin
molecules as happens in solution causing the release of trypsin
peptides. Trypsin autolysis is a common problem that further
complicates the interpretation of mass spectra of the peptides of
interest. By this means, in a single spin, the gel can be
subdivided by the mesh material, which will greatly shorten the
time period required to elute the proteins and cleave them into a
pure aliquot of peptides ready for analysis.
[0090] The present invention also provides other processing steps
for the subdivided gel. For instance, it is also commonplace to
desalt peptides before introducing them into the mass spectrometer.
One particularly convenient way to do this is by absorption of the
peptides onto reversed-phase resins such as C-18 derivatized
silica. This technique is used in a commercial product made by
Millipore Corporation called the ZIPTIP and in the similar product
made by AmiKa of Gaithersburg, Md. The AmiKa product consists of a
resin containing C-18 chains that is attached to the sides of
pipette tips. The sample is drawn up so that the peptides can bind
to the resin and when the fluid is expelled, the salts are washed
out. The bound peptides can be washed further with water then
eluted with a mixed aqueous, such as an organic solvent for further
analysis.
[0091] In addition to processing gels to recover peptides for
analytical techniques such as spectroscopy (e.g., IR, UV, NMR and
the like) and mass spectrometry, the present invention can be used
to prepare proteins for Edman sequencing, extraction of samples of
nucleic acid for sequencing or amplification, or isolation for use
in protein-protein, nucleic acid-protein or nucleic acid-nucleic
acid interaction studies where intact protein and/or nucleic acid
is needed, and other applications, as would be apparent to one
skilled in the relevant art.
EXAMPLE 3
[0092] In the present invention, a reversed phase layer can be
placed beneath the immobilized trypsin so that the peptides are
captured as they are produced. For example, a layer of C-18
modified glass particles may be placed on a filter membrane as
described above for immobilized trypsin. The particles are
available from Phenomenex, Torrance, Calif.
[0093] The immobilized C-18 membrane, as with the immobilized
trypsin membrane, can be used in series or in a separate reaction
from the subdividing mesh material. In fact, numerous varying
membranes may be stacked in any number of custom sequences that a
researcher may desire. As an example, a first mincing tube
containing a mesh for subdivision of the gel can be placed atop a
second nesting treating tube containing immobilized trypsin on a
membrane. The sample is first centrifuged at a force sufficient to
drive the gel through the mesh and subdivide it but not with a
force sufficient to drive the solution through the membrane which
holds the trypsin in place. Next, the subdivided gel would be
allowed to incubate on the trypsin membrane for time sufficient to
allow the protein to be digested. Techniques and incubation times
for trypsin digestion of protein are well known in the art.
[0094] The tube is then spun to force the peptides and fluid
through the conditionally porous membrane holding the trypsin and
onto a second conditionally porous membrane filter on which C-18
glass particles are trapped. As the fluid flows through this
filter, peptides are captured by the C-18 while the salt solution
flows through to the tube bottom or into a separate recovery
vessel. The fluids are forced through the column of mesh and
membranes having immobilized phases by centrifugal force. A fresh
recovery vessel can be placed below the column and the peptides can
be eluted into it by applying an elution solvent or buffer of
appropriate strength to the top of the column and centrifuging it a
second time.
[0095] Yet another embodiment uses a nested set of treatment and
purification techniques with immobilized antibodies to capture and
remove from an analyte sample proteins that are not of interest.
For example, it is quite common that after gel separation of
proteins one finds that certain high abundance "housekeeping"
proteins existing in substantial quantities, migrate in the gel to
the same location as low abundance proteins of interest. The high
abundance proteins mask the low abundance proteins and make their
identification or sequencing difficult. One way to solve this
problem is to remove the high abundance proteins, which are
generally well known and easily isolated, by binding them
selectively with monoclonal antibodies or with polyclonal
antibodies raised against them. Methods for the preparation of
antibodies are well known in the art.
[0096] Although Example 3 describes the use of a membrane having
C-18 particles for capturing proteins and peptides for desalting, a
membrane having attached alkyl chains shorter than or longer than
18 carbons (e.g., C6, C8, C10, C12, C20, etc.) could be
successfully used with the invention. Membranes to which long alkyl
chains may be attached are manufactured by Pall Gelman Corp. of Ann
Arbor, Mich. Restek Corp. of Bellefonte, Pa. sells extraction disks
having alkyl chains of either C-8 or C-18 bound to glass fibers.
(C18 disk, 47 mm, Cat. #24004; C8 disk, 47 mm, Cat. #24048). These
could also be used as capture media.
[0097] In addition to using a membrane or particle coated with an
alkyl chain to capture peptides, one might use ion exchange resin
particles or, alternatively, membranes modified to have ion
exchange properties--derivatized with ionizable groups. Such ion
exchangers might be very effective, for example, to capture nucleic
acids or certain highly ionic peptides or proteins.
EXAMPLE 4
[0098] In this example, antibodies are immobilized on activated
glass or other similar substrate in the same way as the trypsin
described above. Particles with attached antibodies are placed atop
a filter membrane or other similar support with pores sufficient to
retain the particles, or the antibodies are immobilized to the
membrane itself or to a material, which will not penetrate the
filter membrane such as glass fiber. The membrane is then secured
across the opening of the tube as previously described. When
protein or similar mixtures are placed atop the membrane and the
whole assembly centrifuged, the component parts of the mixture
capable of binding to the immobilized antibodies are captured and
retained above the filter membrane. The filtrate is thus depleted
of high abundance proteins, thus allowing the lower abundance
proteins to be examined in further analytical procedures.
[0099] Particularly useful in this embodiment is a filter membrane
that does not readily admit passage of the solutions until
subjected to centrifugal force, or, a conditionally porous
membrane. Such a membrane could be a polycarbonate membrane that is
commercially available from Poretics Corporation of Livermore,
Calif.
EXAMPLE 5
[0100] The invention can be used and adapted for use with standard
processing techniques, such as dehydration and rehydration recovery
techniques. One example, using the invention in an In-Gel Tryptic
Digest Protocol is described below. This protocol could be varied,
as would be apparent to one skilled in the relevant art. The steps
are as follows:
[0101] 1. Excise a desired band from a gel using a clean scalpel or
similar excision tool;
[0102] 2. Place the gel in a nested tube on top of a mesh
material;
[0103] 3. Spin the tube at 10,000 rpm to mince the gel and draw the
subdivided gel fragments into a second nested receptacle containing
a conditionally porous membrane;
[0104] 4. Add 250 .mu.L of 50% H.sub.2O/50% acetonitrile wash to
the minced gel and wash for 5 minutes;
[0105] 5. Remove the acetonitrile wash by centrifuging the solution
through the membrane;
[0106] 6. Add 250 .mu.L 50% acetonitrile/50 mM NH.sub.4HCO.sub.3
solution and wash for 30 minutes at room temp, agitation may be
helpful;
[0107] 7. Remove the acetonitrile/NH.sub.4HCO.sub.3 solution wash
by centrifuging;
[0108] 8. Add 250 .mu.L 50% acetonitrile/10 mM NH.sub.4HCO.sub.3
and wash for 30 minutes at room temperature;
[0109] 9. Spin to remove the acetonitrile/NH.sub.4HCO.sub.3
solution and discard the solutions;
[0110] 10. Speedvac the minced gel fragments to complete
dryness;
[0111] 11. Add 0.1 .mu.g modified trypsin (Promega) per 15 mm.sup.3
of gel in 15 .mu.L of 10 mM NH.sub.4HCO.sub.3;
[0112] 12. Let stand for 5-10 minutes to allow enzyme/buffer
solution to absorb into the gel;
[0113] 13. Add an additional 20 .mu.L of 10 mM NH.sub.4HCO.sub.3
buffer without any additional enzyme;
[0114] 14. Verify that all the minced gel fragments are covered
with the buffer, and if not, increase the buffer volume to cover
all the gel fragments;
[0115] 15. Incubate at 37.degree. C. for 24 hours;
[0116] 16. Centrifuge and collect supernatant;
[0117] 17. Perform a second extraction using 200 .mu.L of 60%
acetonitrile in water containing 0.1% TFA, and shaking at room
temperature for 60 minutes;
[0118] 18. Centrifuge to collect the extract solution from the
receiver tube;
[0119] 19. Generally two extracts are all that is need for protein
identification by MALDI-TOF MS, however, for quantitation purposes,
a third extraction may be necessary and thus steps 17 and 18 would
be repeated;
[0120] 20. Speedvac the extract solution down to 5-10 .mu.L;
and
[0121] 21. For MALDI-MS mix 1 .mu.L of the extract with 1 .mu.L of
internal standards (i.e. containing 100 fmol of bradykinin) and 1
.mu.L of .alpha.-cyano-4-hydroxy-trans-cinnamic acid.
EXAMPLE 6
[0122] An additional sample protocol using the present invention is
described below.
[0123] 1. Excise both a desired gel band and a blank gel piece of
approximately the same size with an ethanol rinsed scalpel;
[0124] 2. Place each sliced gel on top of a mesh material in
individual mincer tubes respectively;
[0125] 3. Spin tubes at 10,000 rpm to mince the gels and pass gel
fragments onto membranes;
[0126] 4. Dehydrate the minced fragments in 200 .mu.L MeOH for 5
minutes, then rehydrate the fragments with 200 .mu.L 30%
MeOH/H.sub.2O, shaking the tube for 5 minutes;
[0127] 5. Wash gel fragments in 200 .mu.L water for 10 minutes, two
times;
[0128] 6. Wash gels fragments with 100 mM ammonium bicarbonate/30%
acetonitrile for 10 minutes at a time, until colorless (minimum 3
washes). One final wash in water may be done to reduce buffer
concentration and speed the drying process;
[0129] 7. Dry the gel fragments in a speedvac;
[0130] 8. Rehydrate the gel fragments in 2 mM Tris-HCl/300 ng
modified sequence grade Trypsin or 25 mM ammonium bicarbonate/300
ng modified sequence grade Trypsin, taking care to provide
sufficient volume to completely cover the gel fragments in case
they swell;
[0131] 9. Carefully vortex to mix the gel fragments and rehydration
solution and incubate at 37.degree. C. for at least 8 hours;
[0132] 10 Centrifuge the tube to pass the supernatant to the bottom
of the tube;
[0133] 11. Extract fragments using 50% acetonitrile/0.1% TFA for 10
minutes at room temp;
[0134] 12. Centrifuge to add extract solution to the supernatant
from the digestion;
[0135] 13. Speedvac the extract solution to reduce the solution
volume to 10 .mu.L; and
[0136] 14. Apply 1 .mu.L of the extract with 2 .mu.L of the
appropriate MALDI matrix for MS analysis.
[0137] The protocol may be varied in many ways, as would be
apparent to one skilled in the relevant art. Furthermore,
additional or optional treatments or steps may be used. For
instance, in one optional treatment, the centrifuge tube may
contain a few milligrams of reverse phase resin. After collecting
the supernatant and first extract as described in step 9-11, the
volume of the acetonitrile solution may be reduced to allow binding
of the digested peptides to the resin. The resin then, may be
washed several times with 25 mM ammonium bicarbonate. Peptides may
be removed from the resin by mixing the resin with 80%
acetonitrile/25 mM ammonium bicarbonate. The eluted peptides are
transferred to a clean microfuge tube and evaporated to 10 .mu.L,
using a speedvac.
EXAMPLE 7
[0138] An additional sample protocol using the present invention is
described below.
[0139] 1. Excise a desired band from a gel using a clean scalpel or
similar excision tool;
[0140] 2. Place the gel slice in a nested tube on top of a mesh
material;
[0141] 3. Spin tube at 10,000 rpm to mince gel and draw minced gel
fragments onto a membrane when using an Eppendorf 5415C
centrifuge;
[0142] 4. Remove first nested tube containing the mincing mesh;
[0143] 5. Wash gel by adding 100 .mu.L of ultra pure water to cover
the minced gel pieces. If pieces are not covered, add enough water
to completely cover the fragments;
[0144] 6. Allow the gel to incubate for 15 minutes, then spin the
tube to pass the water through the membrane and into the collection
tube;
[0145] 7. Repeat steps 5 and 6;
[0146] 8. Repeat steps 5 and 6 using 25 mM ammonium bicarbonate
instead of ultrapure water;
[0147] 9. Transfer the nested tube containing the membrane and
minced gel pieces into a clean 1.5 mL Eppendorf microfuge tube and
place into a speedvac;
[0148] 10. Speedvac the gel fragments to complete dryness;
[0149] 11. Add 0.1 .mu.g of modified trypsin (Promega) per 15
mm.sup.3 of gel in 25 .mu.L of 10 mM ammonium bicarbonate;
[0150] 12. Let stand for 5-10 minutes to allow the enzyme/buffer
solution to absorb into the gel;
[0151] 13. Add an additional 25-50 .mu.L of ammonium bicarbonate
without enzyme to completely cover all of the gel fragments;
[0152] 14. Incubate at 37.degree. C. overnight or approximately 16
hours;
[0153] 15. Centrifuge and collect the supernatant;
[0154] 16. Remove the nested tube containing the membrane and gel
fragments from the Eppendorf collection tube and reduce the volume
to 5-10 .mu.L using a speedvac (the first extract typically
contains enough peptide fragments for protein identification by
MALDI-TOF Mass spectrometry); and
[0155] 17. Apply the collected solution containing peptides
fragments, along with the appropriate MALDI matrix, to the stage
plate and an internal standard, if desired.
EXAMPLE 8
[0156] In one aspect of the invention, an Eppendorf 5415C
centrifuge is used to mince a gel. Test results are displayed in
the table below.
1 TABLE 1 10% 10% 12% 20% 4% TG 8% TG TG BT BT TG RCF A B A B A B A
B A B A B 2040 .times. g - + - - 4000 .times. g + + - + 8160
.times. g + + +/- + +/- +/- 11,750 .times. g + + + + +/- + +/- +
16,000 .times. g + + +/-
[0157] The gels used in the test and the symbol definitions are set
forth below.
[0158] 4% TG--4% acrylamide crosslinked with 2.5% bisacrylamide
[0159] 8% TG--8% acrylamide crosslinked with 2.5% bisacrylamide
[0160] 10% TG--10% acrylamide crosslinked with 2.5%
bisacrylamide
[0161] 10% BT--10% acrylamide crosslinked with 4.1%
bisacrylamide
[0162] 12% BT--12% acrylamide crosslinked with 5% bisacrylamide
[0163] 20% TG--20% acrylamide crosslinked with 2.5%
bisacrylamide
[0164] "A" represents a mesh material having openings with a width
of 0.0145 inch, and a wire diameter 0.0055 inch.
[0165] "B" represents a mesh material having openings with a width
of 0.0267 inch, and wire diameter of 0.0065 inch.
[0166] "+" represents gel completely drawn through the mesh.
[0167] "-" represents gel not drawn through the mesh.
[0168] "+/-" represents some of the gel drawn through the mesh and
some of the gel left on the mesh.
[0169] In specific embodiments, centrifuging to facilitate passage
of the semisolid material through the mesh, thereby subdividing the
semisolid material into fragments, can be completed at Relative
Centrifugal Force (RCF) in the ranges of about 1,000.times.g to
26,000.times.g, about 1,000.times.g to 24,000.times.g, about
1,000.times.g to 20,000.times.g, about 1,000.times.g to
16,000.times.g, about 1,000.times.g to 12,000.times.g, about
1,000.times.g to 8,000.times.g, about 1,000.times.g to
6,000.times.g, about 1,000.times.g to 4,000.times.g, about
1,000.times.g to 2,000.times.g, about 4,000.times.g to
24,000.times.g, about 4,000.times.g to 20,000.times.g, about
4,000.times.g to 16,000.times.g, about 4,000.times.g to
12,000.times.g, about 4,000.times.g to 8,000.times.g, about
4,000.times.g to 6,000.times.g, about 8,000.times.g to
24,000.times.g, about 8,000.times.g to 20,000.times.g, about
8,000.times.g to 16,000.times.g, about 8,000.times.g to
12,000.times.g about 8,000.times.g to 10,000.times.g, about
10,000.times.g to 24,000.times.g, about 10,000.times.g to
20,000.times.g, about 10,000.times.g to 16,000.times.g, about
10,000.times.g to 12,000.times.g, about 12,000.times.g to
24,000.times.g, about 12,000.times.g to 16,000.times.g, about
12,000.times.g to 14,000.times.g, about 16,000.times.g to
24,000.times.g, about 16,000.times.g to 20,000.times.g and about
20,000.times.g to 24,000.times.g.
[0170] The use of centrifugal force to drive the solutions through
the various layers, affixes the layers together. Thus, a pressure
seal between the layers is optional. Each tube segment is an
interchangeable module so that the user can readily assemble a
nested tube set custom designed for his particular analyte
processing needs.
[0171] For example, one part of the nested set can provide the
sieve or mesh material to subdivide the gel. Another part can
provide the immobilized trypsin held in place by a porous membrane
or conditionlly porous membrane or immobilized on a membrane
itself. Another part can provide the reversed phase capture
material, either held in place by a membrane or by using membrane
derived so as to bind proteins by hydrophobic interactions. Another
part can provide the immobilized antibody to capture a high
abundance protein. Several types of such immobilized antibodies (or
portions thereof, e.g., Fc fragments, Fab or Fab'.sub.2 fragments,
H or L chains, or combinations thereof) might be provided either as
separate segments for nesting or combined in a single segment. The
last segment can be a receptacle or recovery vessel for fluid
driven through the column by centrifugal force. Such a segment can
be removed to discard fluids and a fresh one put in place for the
elution of the peptides or proteins of interest from the column.
Moreover, segments of a nested tube set can be removed between
steps.
[0172] Specifically, if the objective is to capture peptides on a
reversed phase packing after the gel is subdivided by the mesh and
digested with trypsin, the mesh and trypsin segments of this nested
set of tubes might be removed and discarded or cleaned for reuse
before the peptides are eluted from the reversed phase with a mixed
solvent system.
[0173] As a further example of the versatility of this invention,
tubes may be inverted and placed into larger filter-containing
tubes for subsequent processing steps. For example, following
subdivision of gel and extraction of the protein, if some of the
more abundant proteins need to be removed, a second tube containing
a filter and antibodies immobilized on membrane can be used. The
second tube is placed on the top in way that when the tubes are
inverted and spun briefly by the centrifuge, the sample containing
solution passes through the filter and comes in contact with the
immobilized antibodies. The protein can be kept in contact with the
antibodies as long as needed. This same concept of inversion can be
extended to the other sample processing steps such as digestion,
purification and so on.
[0174] The system for mincing and treating gel materials is highly
flexible since a wide variety of processes can be conducted in
series in the nested tubes and different procedures can be selected
by using different prepared tubes. For example, one tube might
contain trypsin while another might contain papain, and the user
can select whichever tube best meets the needs of the process to be
conducted. In one embodiment, these nested tubes are nested
microtiter plates, wherein an array of nested tubes align with
tubes of a microtiter plate. A microtiter plate links together, or
has formed therein, an array of tubes with closed bottoms. A
mincing tube or a treating tube could be a set of tubes arrayed in
an 8.times.12 or 16.times.24 format, or other format, with centers
compatible with standard microtiter plates so that the tube array
can be mated with conventional microtiter plates as receiver
plates. Since centrifuge equipment is readily available from Fisher
(Marathon 21000 and 21000R centrifuges, Fisher Scientific,
Pittsburgh, Pa.), Brinkmann (Eppendorf 5804 and 5810 centrifuges,
Brinkmann Instruments Inc., Westbury, N.Y.) and others, that allow
an entire microtiter plate of 96, 384 or more wells to be spun
simultaneously, the above-described nested tube set can be provided
in the form of nested microtiter plates. This will allow easy
simultaneous processing of a multiplicity of samples without having
to handle individual tube segments, making this invention
especially useful in high throughput analysis.
[0175] The invention further includes methods for isolating and/or
purifying molecules such as macromolecules, peptides,
oligonucleotides, and carbohydrates. These methods comprise (a)
placing a gel subportion upon the mesh material of a mincing tube
described above, (b) centrifuging the mincing tube, the mesh, and
the gel subportion to facilitate passage of the gel subportion
through the mesh so as to divide the gel subportion into fragments,
and (c) extracting the gel subportion fragments with an extraction
solution. Optionally, the molecules extracted by the extraction
solution, and present therein, may be concentrated by, for example,
precipitation or evaporation of extraction solution solvent(s).
Typically, the gel subportion fragments will be separated from the
extraction solution prior to analysis by mass spectroscopy.
[0176] The invention also includes methods for isolating and/or
purifying molecules such as macromolecules, peptides,
oligonucleotides, and carbohydrates. These methods comprise (a)
separating one or more molecules by gel electrophoresis, (b)
sectioning the resulting gel to obtain a subportion thereof which
contains one or more molecules of interest, (c) placing the gel
subportion upon the mesh material of a mincing tube described
above, (d) centrifuging the mincing tube, the mesh, and the gel
subportion to facilitate passage of the gel subportion through the
mesh so as to divide the gel subportion into fragments, and (e)
extracting the gel subportion fragments with an extraction
solution.
[0177] As one skilled in the art would recognize, extraction
solutions used in methods of the invention may be either aqueous or
non-aqueous.
[0178] Further, the pH of extraction solutions used in methods of
the invention may be basic, acidic, or neutral. When basic
extraction solutions are used, these solutions may have a pH which
is about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about
10.0, about 10.5, about 11.0, about 11.5, about 12.0, about 12.5,
about 13.0, about 13.5, or about 14.0. When acidic extraction
solutions are used, these solutions may have a pH which is about
6.5, about 6.0, about 5.5, about 5.0, about 4.5, about 4.0, about
3.5, about 3.0, about 2.5, about 2.0, about 1.5, about 1.0, or
about 0.5. Further, the extraction solution may have a pH in any of
the following ranges: from about pH 1.0 to about pH 5.0, from about
pH 2.0 to about pH 6.0, from about pH 3.0 to about pH 7.0, from
about pH 4.0 to about pH 8.0, from about pH 5.0 to about pH 9.0,
from about pH 6.0 to about pH 10.0, from about pH 7.0 to about pH
11.0, from about pH 8.0 to about pH 12.0, and from about pH 9.0 to
about pH 13.0.
[0179] Extraction solutions used in methods of the invention may
contain one or more agents which facilitate and/or inhibit the
degradation of particular classes of molecules. For example, when
methods of the invention are designed for the purification and/or
isolation of proteins, the extraction solution may contain protease
inhibitors (e.g., PMSF) and one or more ribonucleases which will
facilitate the digestion of RNA. Similarly, when methods of the
invention are designed for the purification and/or isolation of
DNA, the extraction solution may be substantially free of
deoxyribonucleases but, may contain one or more ribonucleases.
[0180] The invention further includes methods for identifying
molecules such as macromolecules, peptides, oligonucleotides, and
carbohydrates. These methods comprise (a) placing a gel subportion
upon the mesh material of a mincing tube described above, (b)
centrifuging the mincing tube, the mesh, and the gel subportion to
facilitate passage of the gel subportion through the mesh so as to
divide the gel subportion into fragments, (c) extracting the
molecules from the gel subportion fragments with an extraction
solution, and (d) performing mass spectroscopy (e.g., tandem mass
spectroscopy, matrix-assisted laser desorption/ionization mass
spectrometry (MALDI-MS), inductively coupled plasma mass
spectroscopy (ICP-MS), Fourier transform ion cyclotron resonance
mass spectroscopy, (FTICR-MS), electrospray mass spectrometry
(ES-MS), etc.) on the extraction solution to identify molecules
present. Typically, the gel subportion fragments will be separated
from the extraction solution prior to analysis by mass
spectroscopy.
[0181] The invention also includes methods for identifying
molecules such as macromolecules, peptides, oligonucleotides, and
carbohydrates. These methods comprise (a) separating one or more
molecules by gel electrophoresis, (b) sectioning the resulting gel
to obtain a subportion thereof which contains one or more molecules
of interest, (c) placing the gel subportion upon the mesh material
of a mincing tube described above, (d) centrifuging the mincing
tube, the mesh, and the gel subportion to facilitate passage of the
gel subportion through the mesh so as to divide the gel subportion
into fragments, (e) extracting the molecules from the gel
subportion fragments with an extraction solution, and (f)
performing mass spectroscopy (or other analytical techniques, such
as those described elsewhere herein) on the extraction solution to
identify molecules present.
[0182] The invention further includes methods for sequencing
molecules such as macromolecules, peptides, oligonucleotides, and
carbohydrates. These methods comprise (a) placing a gel subportion
upon the mesh material of a mincing tube described above, (b)
centrifuging the mincing tube, the mesh, and the gel subportion to
facilitate passage of the gel subportion through the mesh so as to
divide the gel subportion into fragments, (c) extracting the
molecules from the gel subportion fragments with an extraction
solution, and (d) performing mass spectroscopy (e.g., tandem mass
spectroscopy, MALDI-MS, ES-MS, FTICR-MS, ICP-MS, etc.) on the
extraction solution to identify molecules present. As noted above,
typically, the gel subportion fragments will be separated from the
extraction solution prior to analysis by mass spectroscopy.
[0183] The invention also includes methods for sequencing molecules
such as macromolecules, peptides, oligonucleotides, and
carbohydrates. These methods comprise (a) separating one or more
molecules by gel electrophoresis, (b) sectioning the resulting gel
to obtain a subportion thereof which contains one or more molecules
of interest, (c) placing the gel subportion upon the mesh material
of a mincing tube described above, (d) centrifuging the mincing
tube, the mesh, and the gel subportion to facilitate passage of the
gel subportion through the mesh so as to divide the gel subportion
into fragments, (e) extracting the molecules from the gel
subportion fragments with an extraction solution, and (f)
performing mass spectroscopy (e.g., tandem mass spectroscopy,
MALDI-MS, ES-MS, FTICR-MS, ICP-MS, etc.) on the extraction solution
to identify molecules present.
[0184] Methods for performing mass spectroscopy are described in
U.S. Pat. Nos. 6,238,871, 5,955,729, 5,854,486, 5,716,825,
5,705,813, the entire disclosures of which are incorporated herein
by reference. In particular, Koster et al., U.S. Pat. No.
6,238,871, describe methods for sequencing nucleic acids using mass
spectrometry (e.g., MALDI-MS and ES-MS).
[0185] It should be understood, that the described methods may be
used to isolate proteins, peptides, nucleic acids and other
substances, including all types of macromolecules.
[0186] In one aspect of the invention, a kit for mincing and
treating a gel is provided. Kits serve to expedite the performance
of, for example, methods of the invention by providing multiple
components and reagents packaged together. Further, reagents of
these kits can be supplied in pre-measured units so as to increase
precision and reliability of the methods. The kit may comprise a
carrier being compartmentalized to receive one or more components
of the kit. Kits of the invention for mincing a gel and extracting
an analyte from a gel generally comprising a carton such as a box,
one or more containers such as boxes, tubes, ampules, jars, bags,
plates and the like, a mincing tube and any combination of one or
more of the below listed items.
[0187] The kit of the invention may comprise a receiving vessel or
a treating tube containing a conditionally porous membrane, as
described above with reference to the Figures.
[0188] The kit may comprise one or more solutions, or a material to
prepare one or more solutions, to treat a gel, such as, for
example, a trypsin enzyme in a compatible salt solution or,
alternatively, a packaged dry enzyme. The solution may include an
aqueous, nonaqueous or mixed solvent for eluting captured proteins
or peptides from a conditionally porous membrane. Further, the
solution could be a solvent, a buffer solution, or a solution
containing a reagent, such as trypsin, a long-chain alkyl group, an
ion exchange group, a short chain carboxylate or sulfonate, a
chelating group or a boronic acid. The kit may include other
solutions including aqueous, nonaqueous or mixed solvents to be
used in eluting captured proteins or peptides from conditionally
porous membranes which have been modified to treat or extract
components from the gel.
[0189] The kit may include one or more spare membranes or spare
mesh material, or mesh material having different hole sizes to
enable a user to custom fragment the gel. The kit may contain
printed instructions for use.
[0190] The kit may comprise one or more treated or untreated
membranes for the selective removal of components from the gel
which may interfere or compromise subsequent analytical procedures.
The membranes may be treated with long-chain alkyl groups, ion
exchange groups such as tertiary or quaternary amines, short chain
carboxylates or sulfonates, chelating groups for metal ion affinity
capture or boronic acid. The kit may include particles, such as ion
exchange or reversed phase capture particles on the membrane.
[0191] The kit of the invention may further comprise one or more
items including a gel or a gel electrophoresis apparatus, a pipette
or a spatula.
[0192] While the invention has been particularly shown and
described with reference to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and detail may be made therein without departing from the
spirit and scope of the invention.
[0193] All publications, articles and patents referred to herein,
including U.S. Provisional Application No. 60/218,821, are
incorporated by reference in their entirety.
* * * * *