U.S. patent application number 12/060834 was filed with the patent office on 2008-07-31 for labeling of immobilized proteins using dipyrrometheneboron difluoride dyes.
This patent application is currently assigned to INVITROGEN CORPORATION. Invention is credited to Richard P. Haugland, Karen J. Martin, Wayne F. Patton.
Application Number | 20080182284 12/060834 |
Document ID | / |
Family ID | 34859785 |
Filed Date | 2008-07-31 |
United States Patent
Application |
20080182284 |
Kind Code |
A1 |
Haugland; Richard P. ; et
al. |
July 31, 2008 |
LABELING OF IMMOBILIZED PROTEINS USING DIPYRROMETHENEBORON
DIFLUORIDE DYES
Abstract
The invention describes methods for labeling or detecting of
immobilized poly(amino acids), including peptides, polypeptides and
proteins, on membranes and other solid supports, using fluorescent
dipyrrometheneboron difluoride dyes. Such immobilized poly(amino
acids) are labeled or detected on blots or on arrays of poly(amino
acids), or are attached to immobilized aptamers.
Inventors: |
Haugland; Richard P.;
(Olympia, WA) ; Martin; Karen J.; (Douglas,
AK) ; Patton; Wayne F.; (Newton, MA) |
Correspondence
Address: |
INVITROGEN CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
INVITROGEN CORPORATION
Carlsbad
CA
|
Family ID: |
34859785 |
Appl. No.: |
12/060834 |
Filed: |
April 1, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11210438 |
Aug 23, 2005 |
|
|
|
12060834 |
|
|
|
|
10005050 |
Dec 3, 2001 |
6972326 |
|
|
11210438 |
|
|
|
|
Current U.S.
Class: |
435/21 ; 435/28;
435/4; 436/518; 436/86 |
Current CPC
Class: |
G01N 33/6839 20130101;
C07K 1/13 20130101; G01N 33/54306 20130101; G01N 33/583 20130101;
C07K 1/1077 20130101; G01N 33/581 20130101; G01N 27/44726
20130101 |
Class at
Publication: |
435/21 ; 436/86;
435/4; 436/518; 435/28 |
International
Class: |
C12Q 1/42 20060101
C12Q001/42; G01N 33/00 20060101 G01N033/00; C12Q 1/00 20060101
C12Q001/00; G01N 33/543 20060101 G01N033/543; C12Q 1/28 20060101
C12Q001/28 |
Claims
1. A kit for detection of poly(amino acids) immobilized on a solid
surface, said kit comprising: a. a dipyrrometheneboron difluoride
dye of the formula: ##STR00002## wherein each of R.sup.1 through
R.sup.7 are independently selected from the group consisting of H,
halogen, -L-Rx, and substituted or unsubstituted C.sub.1-C.sub.6
alkyl, aryl, arylethenyl, arylbutadienyl, and heteroaryl; provided
that one or more of R.sup.1 through R.sup.7 is H, two or more of
R.sup.1 through R.sup.7 is nonhydrogen, and only one of R.sup.1
through R.sup.7 is -L-Rx, where L is a spacer having 1-24
nonhydrogen atoms and Rx is a maleimide or a succinimidyl ester of
a carboxylic acid; such that the dipyrrometheneboron difluoride dye
has an absorption maximum between 495 nm and 640 nm; b. a specific
binding pair member that contains a label and that selectively
binds to a target that is its complementary binding pair.
2. The kit according to claim 1, further comprising a fluorogenic
substrate.
3. The kit according to claim 2, wherein the specific binding pair
member contains a label that is an enzyme and the enzyme is capable
of utilizing the fluorogenic substrate to generate a detectable
optical response.
4. The kit according to claim 1, wherein the specific binding pair
member is an antibody or antibody fragment.
5. The kit according to claim 1, wherein the specific binding pair
member contains a label that is a fluorescent dye.
6. The kit according to claim 1, wherein said specific binding pair
member is a biotin-binding protein.
7. The kit according to claim 6, wherein the biotin-binding protein
is avidin, Neutravidin or streptavidin.
8. The kit according to claim 1, wherein the label is an enzyme
that is a peroxidase or a phosphatase.
9. The kit according to claim 8, wherein the peroxidase is
horseradish peroxidase.
10. The kit according to claim 2, wherein the fluorogenic substrate
is a peroxidase substrate that is a fluorescent tyramide.
11. The kit according to claim 8, wherein the phosphatase is
alkaline phosphatase.
12. The kit according to claim 2, wherein the fluorogenic substrate
is a phosphatase substrate that is a
9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate.
13. The kit according to claim 2, wherein the fluorogenic substrate
is a phosphatase substrate that is a
2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone.
14. The kit according to claim 2, wherein the fluorogenic substrate
is a phosphatase substrate that is ELF 39 reagent.
15. The kit according to claim 1, wherein R.sup.1 is methyl or
-L-Rx; R.sup.2 is H, bromine, or -L-Rx; R.sup.3 is H or methyl;
R.sup.4 is H or -L-Rx; R.sup.5 is H, methyl, or phenyl; R.sup.6 is
H or bromine; and R.sup.7 is methyl, phenyl, alkoxyphenyl,
phenylethenyl, phenylbutatdienyl pyrrolyl, or thienyl; where -L- is
--(CH.sub.2).sub.2--, --(CH.sub.2).sub.4--,
--OCH.sub.2C(O)NH(CH.sub.2).sub.5--,
--(CH.sub.2).sub.2--C(O)NH(CH.sub.2).sub.5--,
--(CH).sub.2C.sub.6H.sub.4OCH.sub.2C(O)NH(CH.sub.2).sub.5--; and Rx
is a succinimidyl ester of a carboxylic acid; and the specific
binding pair member is an antibody or a streptavidin that contains
a label that is an alkaline phosphatase and the fluorogenic
substrate is a 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)
phosphate, a
2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone,
or ELF 39 reagent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
11/210,438 filed Aug. 23, 2005, which is a division of U.S. Ser.
No. 10/005,050, filed Dec. 3, 2001, which disclosures are herein
incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to labeling or detecting of poly(amino
acids), including peptides, polypeptides and proteins, that are
immobilized on membranes and other solid supports, using
dipyrrometheneboron difluoride dyes.
BACKGROUND OF THE INVENTION
[0003] Poly(amino acids) are typically immobilized on membranes and
other solid supports, as in blots or arrays, to enable analysis of
the poly(amino acids). Labeling or detection of such immobilized
poly(amino acids) is of great importance in a multitude of diverse
activities, ranging from basic research to enzyme production,
forensics analysis and diagnostics. When analyzing the immobilized
poly(amino acids), ideally the total poly(amino acid) profile is
labeled in a way that will not interfere with other subsequent
analytical methods that may be directed at specific targets within
the profile.
[0004] When analyzing complex mixtures of proteins and other
poly(amino acids), e.g., mixtures extracted from cells, cell
organelles, tissues, or other biological samples, the first step
involves some method of separating such complex mixtures into
discrete locations or bands, typically by 1-D or 2-D gel
electrophoresis. Proteins in a given band are often identified by
using a specific binding pair member, such as an antibody or
lectin, that binds selectively to the proteins in that band.
However, in order to be accessible to antibodies or other specific
binding pair members, the protein bands must first be transferred
from the gel to a solid support (electroblotted), such as a
membrane. In some cases, the complex mixtures are generated in the
form of arrays that are already immobilized on a solid support, and
the specific binding pair member is used to probe the array of
immobilized proteins or other poly(amino acids) for those having
certain defined characteristics. In addition to the more
traditional arrays of proteins, the discovery of the SELEX.RTM.
(Systematic Evolution of Ligands by EXponential enrichment) process
enabled the identification of nucleic acid-based ligands, referred
to as aptamers, that recognize molecules other than nucleic acids,
including proteins, with high affinity and specificity (Ann. Rev.
Biochem. 64, 763 (1995); Chem. Rev. 97, 349 (1997) and U.S. Pat.
No. 5,270,163). In general, arrays of aptamers are bound to a solid
support, and after a mixture of proteins or other poly(amino acids)
is applied to the aptamer array, unbound poly(amino acids) are
removed. The poly(amino acids) that are immobilized by binding to
the aptamer array can be counterstained and probed.
[0005] In all cases, the specific binding pair members that are
used to identify proteins in a band or location of interest can
also be used to ascertain a number of important characteristics of
the protein, including the presence and quantity of the protein in
the band, the molecular weight of the protein, and the efficiency
of protein extraction or separation. Such specific binding pair
members, however, are only useful for the analysis of the proteins
that are selectively bound by the specific binding pair member and
do not yield any information about the proteins in other bands.
Therefore, a counterstain is typically used to detect all the other
proteins in the mixture to provide a frame of reference for the
analysis.
[0006] Counterstains for proteins like Coomassie Brilliant Blue
(CBB), Amido Black, silver nitrate (silver staining), and colloidal
gold are often used as calorimetric counterstains for
electroblotted proteins (Anal. Biochem. 164, 303 (1987)). Although
most of these methods have been in common use for many years, they
suffer from certain limitations. CBB staining is easy to perform
and shows good reproducibility but is not very sensitive and only
allows the detection of the major components within a protein
sample (typically, not more than 200-300 spots on a blot from a
cell extract that may actually contain thousands of individual
proteins). In addition, the calorimetric counterstains such as
Amido Black and CBB bind so tightly to proteins that destaining is
difficult and residual counterstain often blocks epitopes for
subsequent immunodetection (Meth. Mol. Biol. Ch. 35, 313 (1999);
Anal. Biochem. 276, 129 (1999); Anal. Biochem. 202, 100 (1992)).
Silver staining is up to 100.times. more sensitive than CBB
staining, which makes it suitable for the detection of trace
components within a protein sample (the detection limit is
.about.0.1 ng protein), and is still a method of choice for
analytical gels. The major drawbacks to silver staining are the
poor reproducibility, the limited dynamic range, and the fact that
certain proteins stain poorly, negatively or not at all. Silver
staining is also a procedure that is labor intensive and requires
carefully timed steps to achieve reproducibility. Membranes stained
with colloidal gold have also been found to generate heavy
backgrounds after applying chemiluminescence-based immunodetection
methods (Electrophoresis 21, 2196 (2000)), and staining lightly
enough to yield useful results resulted in poor reproducibility as
the endpoint had to be determined subjectively by the practitioner.
Typically, all of these counterstaining procedures require that
replicate blots be run, one stained with a calorimetric detection
technique and the other blot probed for specific proteins using
immunological methods. However, in order to identify proteins, the
two blots have to be aligned. This leads to problems because often
two blots from the same sample may differ in size due to swelling
or shrinking during the transfer and staining procedures and
precise alignment of the replicate blot is difficult (Biotechniques
30, 266, (2001)).
[0007] In contrast, fluorescent counterstains have certain common
advantages over the colored stains, although the performance
characteristics of certain classes of fluorescent counterstains
differ substantially. Generally, the fluorescent counterstains that
have been described provide a greater linear dynamic range for
quantitation and, are easier to visualize on CCD camera-based and
laser scanner-based imaging systems. Moreover, they do not
interfere with photographic documentation of colored reaction
products generated by enzyme-catalyzed detection systems in common
use, as the fluorescent counterstains are not visible upon white
light illumination, except with particularly abundant proteins.
Fluorescent counterstaining of the total protein profiles on
nitrocellulose or PVDF membranes has been described using, e.g.,
2-methoxy-2,4-diphenyl-2(2H)-furanone (MDPF), SYPRO Rose Plus dye,
SYPRO Ruby dye, fluorescamine, fluorescein isothiocyanate (FITC),
dichlorotriazinylaminofluorescein (DTAF), and dansyl chloride
(Electrophoresis 21, 1123 (2000); BioTechniques 28, 944 (2000)). A
method for creating a permanent protein record using MDPF has been
used. However, MDPF is not compatible with laser-based gel scanners
since it does not absorb significantly in the visible region of the
spectrum and its absorbance in the ultraviolet is also less than
that of the preferred visible light-excitable dyes of this
invention (Electrophoresis 21, 1123 (2000); BioTechniques 28, 944
(2000)). In addition, MDPF-labeled blots must be viewed using wet
PVDF membranes, as fluorescence signal decreases 500-fold upon
drying (Electrophoresis 19, 2407 (1998)). The fluorescent metal
chelate dye, SYPRO Ruby protein blot stain, has been successfully
incorporated into immunodetection procedures employing
calorimetric, fluorescent or chemiluminescent detection reagents
(Anal. Biochem. 276, 129 (1999); Electrophoresis 22, 881 (2001);
Electrophoresis 21, 2196, 2208 (2000)). However, staining of
immobilized proteins by SYPRO Rose Plus and SYPRO Ruby protein blot
stains (both of which only bind noncovalently to proteins) is
easily reversible and both dyes are washed away during the blocking
step prior to incubation with antibodies. Thus, they do not provide
a permanent record of total protein on the blot as do the dyes of
this invention. Reactive fluorescent counterstains such as
fluorescamine, FITC, DTAF and dansyl chloride have also been used
to create a permanent fluorescent protein record (Anal. Biochem.
164, 303 (1987); J. Biochem. Biophys. Methods 46, 31 (2000)).
However, these dyes are not very photostable and their fluorescence
tends to be environment (dansyl) and/or pH (fluoresceins)
sensitive. In addition, the linear range of fluorescence is limited
by fluorescence quenching at high concentrations (see Table 3 in
Example 28). The current invention allows for a permanent record of
both total protein and specific protein(s) with high sensitivity on
a single blot and yields significantly greater sensitivity than
that of the previously described fluorescent dyes for total protein
staining, including the fluorescein-based dyes (see Table 3 in
Example 28). Blots can be viewed using standard UV illumination, by
illumination with a xenon-arc source or with a dual-wavelength
laser-based gel scanner. In particular, all of the preferred dyes
of the present invention have absorption maxima at >495 nm and
extinction coefficients of >50,000 cm.sup.-1M.sup.-1. Thus, they
can be excited by the argon-ion laser, by longer-wavelength lasers
and laser diodes and by other common long-wavelength excitation
sources that are typically utilized in the equipment that yields
the most sensitive detection of proteins.
[0008] The current invention has several distinct advantages over
known methods. The current invention uses dipyrrometheneboron
difluoride dyes (e.g., various substituted
4-bora-3a,4a-diaza-s-indacene difluoride dyes sold by Molecular
Probes, Inc. under the trademark BODIPY) as a counterstain for
immobilized poly(amino acids). These dyes have been found to have a
variety of advantageous properties, including high extinction
coefficients, a high fluorescence quantum yield, spectra that are
essentially insensitive to solvent polarity and pH, a narrow
emission bandwidth, resulting in a higher peak intensity than other
dyes such as fluorescein, a greater photostability than fluorescein
in some environments and lack of an ionic charge (U.S. Pat. Nos.
4,774,339; 5,187,288; 5,248,782; U.S. Pat. Nos. 5,274,113;
5,451,663; and MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND
RESEARCH CHEMICALS ("MP HANDBOOK") by Richard P. Haugland, 6.sup.th
Ed., (1996), and its subsequent 7.sup.th edition and 8.sup.th
edition updates issued on CD Rom in November 1999 and May 2001,
respectively, each of which are incorporated by reference).
However, it is known that dipyrrometheneboron difluoride dyes,
including BODIPY dyes, usually show significant fluorescence
quenching when attached to proteins (see, e.g., U.S. Pat. No.
5,719,031; MP HANDBOOK, supra, 6.sup.th edition at p. 14; and Anal
Biochem 251, 144 (1997)), particularly when proteins are labeled
with a large molar excess of the reactive dipyrrometheneboron
difluoride dye. Unexpectedly, it was found that certain
dipyrrometheneboron difluoride dyes that are used to label proteins
or other poly(amino acids) immobilized on a solid surface do not
suffer from this reported quenching phenomenon. Unexpectedly, this
severe quenching was not observed in the present invention when the
proteins were detected following blotting onto a membrane, even
when the proteins were reacted with a vast excess of the reactive
dye. In addition, in contrast to colorimetric counterstaining
methods, the current invention permits simultaneous dichromatic
visualization of a target protein and the remaining proteins in the
profile on a single electroblot. Since the selected
dipyrrometheneboron difluoride counterstains also absorb
significantly in the UV region of the spectrum, blots can be
visualized using a handheld UV lamp or a UV-epi-illumination system
in conjunction with a photographic or CCD camera. These
counterstains also absorb in the visible region of the spectrum,
and they may be detected with a dual-wavelength laser-based gel
scanner using a photomultiplier tube. Unlike some other reactive
dyes that have been used to detect proteins on a solid support, the
blots can be imaged either wet or dry, as the selected
dipyrrometheneboron difluoride counterstains do not fade upon
drying. Furthermore, the use of dipyrrometheneboron difluoride
counterstains does not interfere with the subsequent methods
commonly used to analyze immobilized proteins.
[0009] Thus, there is an unmet need for an easy to use and
sensitive method for the labeling and detection (and optionally
quantitation) of total proteins and other poly(amino acids) on
membranes and other solid supports, that would overcome the
disadvantages and limitations of current methods. Such methods
would be useful in research, forensics, quality control and medical
diagnostics. This invention meets these and other needs.
SUMMARY OF THE INVENTION
[0010] Poly(amino acids) that are immobilized on a solid support
are labeled according to the present invention by incubating the
poly(amino acids) with a labeling mixture that comprises one or
more chemically reactive dipyrrometheneboron difluoride dyes for a
time sufficient for the dye to form a covalent bond with the
poly(amino acids), and then removing any unbound dye by washing the
immobilized poly(amino acids) with a suitable solvent. Typically,
the solid support is made of solvent-resistant materials such as
nylon, poly(vinylidene difluoride) (PVDF), glass, plastic, and
their derivatives. The immobilized poly(amino acids) generally have
a molecular weight between 500 and 200,000 daltons. For best
results, the dye is present in the labeling mixture in a
concentration of 0.01 micromolar to 10 micromolar. Subsequent
addition of a specific binding pair member that binds selectively
to a target or targets within the sample of immobilized poly(amino
acids) is a preferred aspect of the invention.
[0011] In one aspect of the invention, the poly(amino acids) are
separated by gel electrophoresis prior to being transferred to a
solid support for labeling with the dipyrrometheneboron difluoride
dyes. In another aspect of the invention, the poly(amino acids) are
immobilized in arrays on the solid support for labeling with the
dipyrrometheneboron difluoride dyes. In yet another aspect of the
invention, the poly(amino acids) are immobilized by being
selectively bound to their specific aptamers arrayed on a solid
support
[0012] After the immobilized poly(amino acids) are labeled with the
dipyrrometheneboron difluoride dyes, whether blotted from a gel or
formatted in arrays, the unbound dye is removed (typically by
washing, e.g., with a solvent) and the labeled poly(amino acids)
are detected by illuminating the bound dipyrrometheneboron
difluoride dyes to yield a detectable optical response, typically a
fluorescent optical response, and using the detectable optical
response to detect the corresponding poly(amino acids).
Dipyrrometheneboron difluoride dyes having an excitation peak
between 495 nm and 640 nm are particularly useful for the
invention. In one aspect of the invention, following reaction with
the immobilized proteins and washing to remove unconjugated dyes,
the protein conjugates are illuminated for 5 seconds or less.
[0013] In one aspect of the invention, the proteins or other
poly(amino acids) are bound to aptamers that are, in turn,
previously arrayed on a solid support. Aptamers can be used as a
diagnostic or prognostic tool. In one embodiment, arrays of
aptamers are bound to a solid support, and a protein sample is
applied. Unbound protein is washed off, and dipyrrometheneboron
difluoride dyes that covalently label reactive sites in proteins
but not nucleic acids are used to generate protein profiles. The
labeled proteins on aptamers are detected by using an array reader,
which uses the same light sources as are commonly used in
commercially available blot readers.
[0014] In aspects of the invention that include adding a specific
binding pair member that binds selectively to a target or targets
within the immobilized poly(amino acids), the specific binding pair
member is typically a lectin, a biotin-binding protein, an antibody
or an antibody fragment. In one aspect of the invention, a label is
covalently attached to the specific binding pair member.
Alternatively, the specific binding pair member is unlabeled, but a
secondary binding pair member is added, to which secondary binding
pair member a label is covalently attached and which secondary
binding pair member binds selectively to the specific binding pair
member (e.g., the secondary binding pair member is a secondary
antibody and the specific binding pair member is a primary
antibody). The label on the secondary binding pair member or
specific binding pair member is typically a fluorescent dye, a
biotin, a hapten such as digoxigenin, or an enzyme (e.g., a
peroxidase, luciferase, aequorin, glycosidase or phosphatase).
Where the label is an enzyme, a fluorogenic or chemiluminescent
substrate (e.g., a fluorescent tyramide, a polyfluorinated
xanthene, a fluorogenic quinazolinone, DDAO phosphate, luciferin,
coelenterazine or a dioxetane phosphate) is typically added for
detection of the target. Where the target of the specific binding
pair member is a biotinylated protein, a biotin-binding protein
(e.g., an avidin) to which a label is covalently attached is used
for detection of the target. Where the label is a hapten that is
not biotin, an antibody to that hapten is utilized in the detection
scheme, for instance if the hapten is digoxigenin the specific
binding pair member comprises an antibody to digoxigenin. Unbound
specific binding pair members or secondary binding pair members are
removed for best detection results.
[0015] In one further aspect of the invention, the specific binding
pair member is a chemical stain such as a thiol-reactive dye that
recognizes only those proteins that contain thiols, a dye that
selectively reacts with oxidized glycoproteins such as Pro-Q
Emerald 300 (U.S. patent Ser. No. 09/970,215) or Pro-Q Sapphire 365
or Pro-Q Sapphire 488 oligohistidine stains (Molecular Probes,
Inc).
[0016] In a further embodiment of the invention, kits adapted for
the practice of any of the claimed methods are described. Such kits
typically include one or more dipyrrometheneboron difluoride dyes
and one or more specific binding pair members or secondary binding
pair members to which a label is covalently attached. Where the
label is an enzyme that is capable of utilizing a fluorogenic,
chromogenic or chemiluminescent substrate, the appropriate
fluorogenic, chromogenic or chemiluminescent substrate is
included.
[0017] The methods of the invention are broadly applicable to
labeling any poly(amino acid) from any origin. The methods are
especially useful and applicable to labeling poly(amino acids) from
biological material. Such poly(amino acids) may be contained in
cells or cellular materials and will typically be obtained for the
purposes of immunoassaying to determining the presence or location
of a specific target, diagnosing medical conditions, or identifying
disease states. These and other aspects of the invention are
described in more detail below.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0018] In the present invention, a novel method of labeling and/or
detecting immobilized poly(amino acids) is disclosed, and novel
kits for practicing such methods are provided. To facilitate
understanding of the invention, the disclosure of the invention is
organized in sections, as follows. First, a definition section is
provided to define terms and phrases used commonly throughout the
disclosure. This definition section includes a comprehensive
description of the types of immobilized poly(amino acids), solid
supports, and labeled specific binding pair members that can be
used with the invention. The next section describes specific
dipyrrometheneboron difluoride dyes used in labeling the poly(amino
acids) in methods of the invention. The following section describes
methods of the invention for labeling or detecting immobilized
poly(amino acids). This next section is a description of the kits
adapted for practicing the methods of the invention. Then various
applications and expansions of the invention are described; this
description is followed by detailed examples illustrating the
invention.
DEFINITIONS
[0019] To assist in the understanding of the invention, the
following terms, as used herein, are defined below. "Aptamer" means
a nucleic acid that binds selectively to an intended target
molecule ("target," as defined below). This binding interaction
does not encompass standard nucleic acid/nucleic acid hydrogen bond
formation exemplified by Watson-Crick base pair formation (e.g., A
binds to U or T and G bind to C), but encompasses all other types
of non-covalent (or in some cases covalent) binding. Non-limiting
examples of non-covalent binding include hydrogen bond formation,
electrostatic interaction, Van der Waals interaction and
hydrophobic interaction. An aptamer may bind to another molecule by
any or all of these types of interaction, or in some cases by
covalent interaction. Covalent binding of an aptamer to another
molecule may occur where the aptamer or target molecule contains a
chemically reactive or photoreactive moiety. The term "aptamer" or
"specifically binding nucleic acid" refers to a nucleic acid that
is capable of forming a complex with an intended target substance.
"Target-specific" means that the aptamer binds to a target analyte
with a much higher affinity than it binds to contaminating
materials. "Array" means a two-dimensional spatial grouping or an
arrangement of biomolecules. "Binding pair" refers to first and
second molecules that bind selectively to each other. A specific
binding pair member is the first member of the binding pair, and
binds selectively to the second member of the binding pair, which
is its complement or complementary binding pair member. The binding
between the members of a binding pair is typically noncovalent.
"Binding selectively" refers to the situation in which one member
of a specific intra or inter species binding pair will not show any
significant binding to molecules other than its specific intra or
inter species binding partner (e.g., an affinity of about 100-fold
less). Binding is mediated through hydrogen bonding or other
molecular forces. "Detectable label" or "Label" means a chemical
used to facilitate identification and/or quantitation of a target
substance. Illustrative labels include labels that can be directly
observed or measured or indirectly observed or measured. Such
labels include, but are not limited to, radiolabels that can be
measured with radiation-counting devices; pigments, dyes or other
chromogens that can be visually observed or measured with a
spectrophotometer; chemiluminescent labels that can be measured by
a photomultiplier-based instrument or photographic film, spin
labels that can be measured with a spin label analyzer; and
fluorescent moieties, where the output signal is generated by the
excitation of a suitable molecular adduct and that can be
visualized by excitation with light that is absorbed by the dye or
can be measured with standard fluorometers or imaging systems, for
example. The label can be a luminescent substance such as a
phosphor or fluorogen; a bioluminescent substance; a
chemiluminescent substance, where the output signal is generated by
chemical modification of the signal compound; a metal-containing
substance; or an enzyme, where there occurs an enzyme-dependent
secondary generation of signal, such as the formation of a colored
product from a colorless substrate or a spontaneously
chemiluminescent product from a suitable precursor. The term label
can also refer to a "tag" or hapten that can bind selectively to a
labeled molecule such that the labeled molecule, when added
subsequently, is used to generate a detectable signal. For
instance, one can use biotin as a tag and then use an avidin or
streptavidin conjugate of horseradish peroxidase (HRP) to bind to
the tag, and then use a chromogenic substrate (e.g.,
tetramethylbenzidine) or a fluorogenic substrate such as Amplex
Gold reagent, or a fluorescent tyramide (Molecular Probes, Inc.) to
detect the presence of HRP. In a similar fashion, the tag can be a
hapten or antigen (e.g., digoxigenin or an oligohistidine), and an
enzymatically, fluorescently, or radioactively labeled antibody can
be used to bind to the tag. Numerous labels are known by those of
skill in the art and include, but are not limited to,
microparticles, fluorescent dyes, haptens, enzymes and their
chromogenic, fluorogenic and chemiluminescent substrates and other
labels that are described in the MOLECULAR PROBES HANDBOOK OF
FLUORESCENT PROBES AND RESEARCH CHEMICALS by Richard P. Haugland,
6.sup.th Ed., (1996), and its subsequent 7.sup.th edition and
8.sup.th edition updates issued on CD Rom in November 1999 and May
2001, respectively, the contents of which are incorporated by
reference, and in other published sources. "Counterstain" (as a
verb) means to label all or substantially all of the poly(amino
acids) that are present in the immobilized poly(amino acids).
"Counterstain" (as a noun) means a reagent or combination of
reagents that labels all or substantially all of the poly(amino
acids) that are present in the immobilized poly(amino acids).
"Detectable response" means a change in, or occurrence of, a signal
that is detectable either by observation or instrumentally.
Typically the detectable response is an optical response resulting
in a change in the wavelength distribution patterns or intensity of
absorbance or fluorescence or a change in light scatter,
fluorescence lifetime, fluorescence polarization, or a combination
of the above parameters. Other detectable responses include, for
example, chemiluminescence, phosphorescence, radiation from
radioisotopes, attraction to a magnet and electron density.
"Dipyrromethenoboron difluoride reactive dyes" means any of
chemically reactive derivatives of the dyes described in U.S. Pat.
Nos. 4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,451,663 that
have an absorption maximum below 640 nm. Many dipyrrometheneboron
difluoride reactive dyes are commercially available under the
trademark BODIPY (Molecular Probes, Inc.). "Chemically reactive
derivatives" means those dye derivatives that covalently label
materials under mild conditions. "Chemiluminescent substrate" is
defined as any compound which enters into a chemical reaction with
a peroxide or a phosphatase component to produce chemiluminescence.
Typically chemiluminescence is initiated upon an event, such as
cleavage of a bond which generates an unstable intermediate that
fragments and releases light or a photon as part of the high-energy
state decay process (U.S. Pat. Nos. 4,931,223 and 4,962,192). "Gel
electrophoresis" as used herein, refers to a variety of gels that
may be used in the technique of gel electrophoresis and includes
gels formed by a variety of gel matrix materials, including
polyacrylamide, agarose, polyacrylamide-agarose composites, and the
like. It also encompasses the net migration of a solute under the
influence of an electric field by the combined effects of
electroosmosis and electrophoresis. It further includes any
non-denaturing cathodic or anodic electrophoresis, including sodium
dodecyl sulfate (SDS)-gel electrophoresis, isoelectric focusing and
gradient gels. "Enzyme" means a protein molecule produced by living
organisms, or through chemical modification of a natural protein
molecule, that catalyses chemical reactions of other substances
without itself being destroyed or altered upon completion of the
reactions. Examples of other substances, include but are not
limited to chemiluminescent, chromogenic, or fluorogenic
substances. The term "fluorescent substrate" is used to describe a
substrate that will produce a fluorescent product upon
modification. "Immobilized" means attached to or operatively
associated with an insoluble and/or dehydrated substance or solid
phase that comprises or is attached to a solid support. "Kit" means
a packaged set of related components, typically one or more
compounds or compositions. "Poly(amino acid)" refers to various
natural or synthetic compounds containing molecules of amino acids
of one or more amino substituents linked by the carboxyl group of
one amino acid and the amino group of another, including but not
limited to proteins, polypeptides, glycoproteins, etc. Poly(amino
acids) comprise one or more a targets to which an antibody, lectin,
aptamer, protein-detection reagent, or other similar specific
binding pair member can bind. Typically, the sample from which
poly(amino acids) are selected for analysis comprises tissue, a
cell or cells, cell extracts, cell homogenates, purified or
reconstituted proteins, recombinant proteins, bodily and other
biological fluids, viruses or viral particles, prions, subcellular
components or synthesized proteins. Possible sources of cellular
material for such samples include without limitation plants,
animals, fungi, protists, bacteria, archae, or cell lines derived
from such organisms, including animal cells or animal cell lines
that are normal or diseased. "Selective protein-staining
techniques" means to generate one or more separately detectable
signals for a specific target within the immobilized poly(amino
acid) sample. "Solid support" means a substrate material having a
rigid or semi-rigid surface. Typically, at least one surface of the
substrate will be substantially flat, although it may be desirable
to physically separate certain regions with, for example, wells,
raised regions, etched trenches, or other such topology. Solid
support materials also include spheres (including microspheres),
rods (such as optical fibers) and fabricated and irregularly shaped
items. Solid support materials include any materials that are used
as affinity matrices or supports for chemical and biological
molecule syntheses and analyses, such as, but are not limited to:
poly(vinylidene difluoride) (PVDF), polystyrene, polycarbonate,
polypropylene, nylon, glass, dextran, chitin, sand, pumice,
polytetrafluoroethylene, agarose, polysaccharides, dendrimers,
buckyballs, polyacrylamide, Kieselguhr-polyacrylamide non-covalent
composite, polystyrene-polyacrylamide covalent composite,
polystyrene-PEG [poly(ethylene glycol)] composite, silicon, rubber,
and other materials used as supports for solid phase syntheses,
affinity separations and purifications, hybridization reactions,
immunoassays and other such applications. The solid support may be
particulate or may be in the form of a continuous surface, such as
a microtiter dish or well, a glass slide, a silicon chip, a
nitrocellulose sheet, nylon mesh, or other such materials. "Solvent
resistant material" means a solid support that is insoluble and
inert to solvents commonly utilized in the field of proteomics. The
solid support would preferably be inert to solvents that include
but are not limited to dimethylformamide, methanol, ethanol,
propanol, methylene chloride and N-methyl-2-pyrrolidone. "Target"
means a substance of analytical interest that is analytically
distinguishable from its surrounding environment. "Washing" means
contacting a material with a solution to remove or dilute a
composition introduced in a previous step. Washing solutions are
typically pure solvents or mixtures of solvents, optionally with
salts or pH-modifying agents that solubilize and/or rinse away some
or all of the composition introduced in the previous step.
Selection of Dipyrrometheneboron Difluoride Dyes
[0020] In this method, immobilized poly(amino acids) are labeled by
incubating the immobilized poly(amino acids) with a labeling
mixture that comprises a reactive dipyrrometheneboron difluoride
dye. An extensive assortment of dipyrrometheneboron difluoride dyes
and the effects of various substituents on their spectral
properties have been described previously (U.S. Pat. Nos.
4,774,339; 5,274,113; 5,187,288; 5,248,782; 5,338,854 and
5,433,896, all incorporated by reference). These dyes differ in
their absorption and emission wavelengths; however, the preferred
dyes have substituents that yield dyes with absorption maxima
between 495 nm and 640 nm and extinction coefficients >50,000
cm.sup.-1M.sup.-1.
[0021] In one aspect of the invention, the dipyrrometheneboron
difluoride dyes have the formula:
##STR00001##
wherein each of R.sup.1 through R.sup.7 are independently selected
from the group consisting of H, halogen, L-Rx, and C.sub.1-C.sub.6
alkyl, aryl, arylethenyl, arylbutadienyl, and heteroaryl. By alkyl
is meant a saturated hydrocarbon chain that is optionally further
substituted by carboxylic acid, sulfonic acid, or halogen. By aryl
is mean an unsaturated 5- or 6-membered hydrocarbon ring. By
heteroaryl is meant an unsaturated 5- or 6-membered ring that
contains one or two heteroatoms. Each aryl or heteroaryl ring is
optionally further substituted by C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 perfluoroalkyl, cyano, halogen, azido, carboxylic
acid, sulfonic acid, or halomethyl. For the preferred dyes, one or
more of R.sup.1 through R.sup.7 is H, two or more of R.sup.1
through R.sup.7 is nonhydrogen, and only one of R.sup.1 through
R.sup.7 is -L-Rx. The element L is a spacer having 1-24 nonhydrogen
atoms selected from the group consisting of C, N, O, P, and S and
is composed of any combination of single, double, triple or
aromatic carbon-carbon bonds, carbon-nitrogen bonds,
nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds,
phosphorus-oxygen bonds, and phosphorus-nitrogen bonds. The element
Rx is a reactive group that is a maleimide or a succinimidyl ester
of a carboxylic acid. In one aspect, -L-Rx is at R.sup.1. In
another aspect, -L-Rx is at R.sup.2. In yet another aspect, -L-Rx
is at R.sup.4. In yet another aspect, R.sup.1 is methyl or -L-Rx.
In a further aspect, R.sup.2 is H, bromine, or -L-Rx. Typically,
R.sup.3 is H or methyl. Typically, R.sup.4 is H or -L-Rx; In
preferred embodiments, R.sup.5 is H, methyl or phenyl. Typically,
R.sup.6 is H or bromine. In preferred embodiments, R.sup.7 is
methyl, phenyl, alkoxyphenyl, phenylethenyl, phenylbutatdienyl
pyrrolyl, or thienyl. Preferred embodiments of -L are where -L- is
--(CH.sub.2).sub.2--, --(CH.sub.2).sub.4--,
--OCH.sub.2C(O)NH(CH.sub.2).sub.5--,
--(CH.sub.2).sub.2--C(O)NH(CH.sub.2).sub.5--, or
--(CH).sub.2C.sub.6H.sub.4OCH.sub.2C(O)NH(CH.sub.2).sub.5--.
Preferably, Rx is a succinimidyl ester of a carboxylic acid.
[0022] Dipyrrometheneboron difluoride dyes that are halogenated are
not preferred since halogenation has been shown to reduce the
fluorescence yield of the dye. Dipyrrometheneboron difluoride dyes
that are substituted at positions 1, 2, 3, 5, 6, and 7 only by
hydrogen atoms or alkyl groups tend to have green to yellow-green
fluorescence and are optimally excited by the 488-nm spectral line
of the argon-ion laser. Substitution of dipyrrometheneboron
difluoride dyes by alkenyl, polyalkenyl, aryl and heteroaryl
moieties or combinations of these substituents causes a red-shift
of both the absorption and emission maxima of the fluorophore,
permitting use of the dyes alone or in combination with other
labels that have contrasting optical properties.
[0023] In a preferred embodiment, the dipyrrometheneboron
difluoride reactive dye is an amine-reactive dye since aliphatic
amines are common to most or all proteins. In a more preferred
embodiment, the dipyrrometheneboron difluoride is a succinimidyl
ester and, in the most preferred embodiments the succinimidyl ester
is separated from the fluorophore by an additional seven-atom
aminohexanoyl ("X") spacer. A number of chemically reactive
dipyrrometheneboron dyes are commercially available under the
trademark BODIPY.RTM. dyes, presently including all of those listed
in Table 1. Properties of these dyes are described by Haugland,
HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (6.sup.th Ed.
Molecular Probes, Inc., Eugene, Oreg., 1996), which is incorporated
by reference. Example 29 provides the procedure used to screen the
dyes represented in Table 1 for their suitability for detecting
total-protein content on a blot.
TABLE-US-00001 TABLE 1 Reactive dipyrrometheneboron difluoride dyes
that are preferred for this invention. Commercial
Dipyrrometheneboron difluoride reactive dyes Product*
6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-
BODIPY FL-X, SE propionyl)amino)hexanoic acid, succinimidyl ester
6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-
BODIPY TMR-X, SE diaza-s-indacene-2-propionyl)amino)hexanoic acid,
succinimidyl ester
6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-
BODIPY TR-X, SE yl)phenoxy)acetyl)amino) hexanoic acid,
succinimidyl ester
6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3- BODIPY
R6G-X, SE propionyl)amino)hexanoic acid, succinimidyl ester
4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
BODIPY R6G, SE acid, succinimidyl ester
6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)
BODIPY 630/650-X, styryloxy)acetyl)aminohexanoic acid, succinimidyl
ester SE
4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
BODIPY 530/550, acid, succinimidyl ester SE
4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic
BODIPY 558/568, acid, succinimidyl ester SE
4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid, BODIPY 564/570, succinimidyl ester SE
4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic
BODIPY 576/589, acid, succinimidyl ester SE
4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-
BODIPY 581/591, indacene-3-propionic acid, succinimidyl ester SE
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-
BODIPY 493, 503, propionic acid, succinimidyl ester SE
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic
BODIPY FL C5, SE acid, succinimidyl ester
6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)
BODIPY 650/665-X, styryloxy)acetyl)aminohexanoic acid, succinimidyl
ester SE
2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- BODIPY
FL Br.sub.2, SE indacene-3-propionic acid, succinimidyl ester
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
BODIPY FL, SE acid, succinimidyl ester *Available as of the filing
date from Molecular Probes, Inc., www.probes.com.
[0024] The dipyrrometheneboron difluoride dye concentration in the
labeling mixture is typically between 1 .mu.M and 100 .mu.M,
preferably between about 5 .mu.M and about 20 .mu.M; more
preferably at least 10 .mu.M.
[0025] The synthesis of dipyrrometheneboron difluoride dyes is well
documented in U.S. Pat. Nos. 4,774,339; 5,274,113; 5,187,288;
5,248,782; 5,338,854 and 5,433,896, all incorporated by reference,
and other publications. Many dyes useful for the invention are
available from Molecular Probes, Inc. (Eugene, Oreg.).
[0026] The dipyrrometheneboron difluoride dye-labeled immobilized
poly(amino acid) are illuminated to give an optical response that
is a fluorescence emission (fluorescence response). Illumination is
by a light source capable of exciting the dipyrrometheneboron
difluoride dye-poly(amino acid) complex, typically at or near the
wavelength of an absorption maximum, such as an ultraviolet
(254-370 nm) or visible (495-640 nm) wavelength emission lamp, an
arc lamp, a laser, or even sunlight or ordinary room light.
Preferably, the sample is excited with a wavelength within 20 nm of
the maximum absorption of the dipyrrometheneboron difluoride
dye-poly(amino acid) conjugate.
[0027] Preferably, the dipyrrometheneboron difluoride
dye-poly(amino acid) complexes possess an absorption maximum
between 480 and 650 nm, more preferably between 495 and 640 nm,
most preferably matching the wavelength of a laser-based
illumination source. The complexes also preferably excite with some
efficiency in the UV at or near 300 nm.
METHODS OF THE INVENTION
[0028] The methods of the invention use chemically reactive
dipyrometheneboron difluoride dyes to accomplish counterstaining
poly(amino acids) that are supported of a solid support, wherein
the solid supports include but are not limited to PVDF,
nitrocellulose, polystyrene, glass and solid supports used in
microarray and aptamer technologies. In one aspect of the
invention, the counterstain is used in conjunction with selective
protein-staining techniques to generate one or more separately
detectable signals.
[0029] In one embodiment, the method of labeling immobilized
poly(amino acids) with dipyrrometheneboron difluoride dyes
comprises the steps of:
[0030] a. separating poly(amino acids) by gel electrophoresis
[0031] b. transferring said separated poly(amino acids) to a solid
support, resulting in immobilized poly(amino acids)
[0032] c. combining said immobilized poly(amino acids) on said
solid support with a labeling mixture that comprises one or more
dipyrrometheneboron difluoride dyes for a sufficient time for the
dyes to form a covalent bond with said poly(amino acids)
[0033] The method optionally comprises a step to remove excess and
unreacted dipyrrometheneboron difluoride dye. Typically, the excess
unreacted dye and any of its decomposition products are removed by
washing.
[0034] The poly(amino acids) that are suitable for staining using
this method include both synthetic and naturally occurring
poly(amino acids), comprising both natural and unnatural amino
acids. The poly(amino acids) of the invention include peptides,
polypeptides and proteins. Poly(amino acids) that are labeled and
analyzed according to the present method optionally incorporate
non-peptide regions (covalently or non-covalently) including lipid
(lipopeptides and lipoproteins), phosphate (phosphopeptides and
phosphoproteins), and/or carbohydrate (glycopeptides and
glycoproteins) regions; or incorporate metal chelates or other
prosthetic groups or non-standard side chains; or are multi-subunit
complexes, or incorporate other organic or biological substances,
such as nucleic acids or cofactors. The poly(amino acids) are
optionally relatively homogeneous or heterogeneous mixtures of
poly(amino acids). Typically, the poly(amino acids) are
proteins.
[0035] In one embodiment of the invention, the poly(amino acids)
are immobilized on a membrane, such as a poly(vinylidene
difluoride) (PVDF) membrane, wherein the poly(amino acids) are
applied to the membrane by blotting, spotting, electroblotting or
other methods.
[0036] In one embodiment of the invention, separated poly(amino
acids) in electrophoretic gels are transferred to a filter membrane
or blot or other solid or semi-solid matrix before being combined
with the labeling mixture. The present method is effective for both
denaturing and non-denaturing gels. Denaturing gels optionally
include a detergent such as SDS or other alkyl sulfonate (e.g.,
0.05%-0.1% SDS). Typically, polyacrylamide or agarose gels are used
for electrophoresis. Commonly used polyacrylamide gels include but
are not limited to Tris-glycine, Tris-tricine, mini- or full-sized
gels. Agarose gels include modified agaroses. Alternatively, the
gel is an isoelectric focusing gel or strip. In addition to
polyacrylamide and agarose gels, suitable electrophoresis gels are
optionally prepared using other polymers, such as HYDROLINK.
Alternatively, the electrophoretic gel is a gradient gel. Useful
electrophoretic gels for the present invention are either prepared
according to standard procedures or are purchased commercially.
[0037] In another embodiment of the method a specific binding pair
member that binds selectively to its complementary member is used
to detect a target or targets within the poly(amino acids) that is
the complementary member. Typically, the specific binding pair
member contains a label, such that when the binding pair member
selectively binds to its complementary member, it forms a
label-specific binding pair-target complex. In one aspect, the
specific binding pair member is covalently labeled with an enzyme,
and the enzyme is capable utilizing a chromogenic, fluorogenic or
chemiluminescent substrate to generate a detectable optical
response. The specific binding pair member that is covalently
labeled with an enzyme then selectively binds to the
target/complementary member to form an enzyme-specific binding
pair-target complex.
[0038] In general, an enzyme-mediated technique use an enzyme
labeled attached to one member of a binding pair or series of
binding pairs as a reagent to detect the complimentary member of
the pair or series. In the simplest case, only the members of one
binding pair are used. One member of the specific binding pair is
the analyte, i.e. substance of analytical interest. An enzyme is
attached to the other (complimentary) member pair, forming a
complimentary complex. The complimentary conjugate attaches to its
complimentary analyte to form a complimentary binding complex. The
complimentary binding complexes include but are not limited to,
antibody-antigen interaction, avidin (streptavidin and derivatives
thereof) and biotin (including desthiobiotin and iminobiotin),
lectins and carbohydrates, immunoglobulins and protein A, G, L or
hybrids thereof. Examples 1-5, 8-10, and 15-22 provide experimental
procedures for these methods but these techniques are well known to
one skilled in the art.
[0039] Alternatively, multiple binding pairs may be sequentially
linked to the target complement in the poly(amino acids), the
specific binding pair member, or to both, resulting in a series of
specific binding pairs interposed between the target and the
detectable enzyme label of the specific binding pair member.
Example 23-25 provides a typical procedure for the use of multiple
binding pairs.
[0040] In a preferred embodiment of this method, an amine-reactive
dipyrrometheneboron difluoride dye selected from Table 2 is used as
a general protein detection reagent. Optionally an enzyme label on
a specific binding pair member that is capable of utilizing a
fluorogenic, chromogenic or chemiluminescent substrate to generate
a detectable optical response is used to detect a specific
immobilized protein or proteins. In a preferred embodiment the
enzyme substrate is a fluorogenic substrate; in the most preferred
embodiments the enzyme substrate yields a detectable fluorescent
product at or near the site of binding of the specific binding pair
member to the specific immobilized protein or proteins. In one
embodiment, the enzyme is alkaline phosphatase or horseradish
peroxidase. In a further embodiment, the alkaline phosphatase or
horseradish peroxidase is covalently bound to an antibody to mouse
IgG or to rabbit IgG or to a lectin. In a further embodiment, the
fluorogenic substrate is
9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO
phosphate), a polyfluorinated xanthine (U.S. Pat. No. 6,162,931,
incorporated by reference), a
2-(2'-phosphoryloxyphenyl)-4(3H)-quinazolinone derivative such as
ELF 97 phosphate or ELF 39 phosphate (U.S. Pat. No. 5,443,986,
incorporated by reference) or a tyramide (U.S. Pat. Nos. 5,196,306;
5,583,001 and 5,731,158, which are incorporated by reference). The
quinazolinone derivatives are commercially available from Molecular
Probes, Inc. under the trademark ELF.RTM.. In a further embodiment
the peroxidase substrate is an Alexa Fluor, Oregon Green, Marina
Blue or biotin-XX tyramide (Molecular Probes, Inc.). In an
additional embodiment, the chemiluminescent substrate is the BOLD
substrate (Intergen), Fluoroblot substrate (Pierce Chemical),
ECL.RTM. substrates (Amersham Biosciences) or CSPD.RTM. substrates
(Applied Biosystems).
[0041] The fluorogenic quinazolinone substrates are combined with
the enzyme-specific binding-pair target-complex under conditions
suitable for the formation of a precipitate. Several variations of
fluorogenic quinazolinone substrates are suitable for this
invention, including ELF 97 and ELF 39 phosphates, blot of which
are described in U.S. Pat. No. 5,443,986, which is incorporated by
reference and are commercially available, as described in the
MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH
CHEMICALS by Richard P. Haugland, 6.sup.th Ed., (1996), and its
subsequent 7.sup.th edition and 8.sup.th edition updates issued on
CD Rom in November 1999 and May 2001, respectively, the contents of
which are incorporated by reference, and in other published
sources. Examples 4, 5, 23, 24, 25 and 32 contain methods relating
to the use of quinazolinone derivatives as a substrate.
[0042] The fluorogenic substrate DDAO phosphate is combined with
the enzyme-specific binding-pair target-complex under conditions
suitable for formation of the precipitate. The DDAO phosphate
substrate (U.S. Pat. No. 4,810,636) is described in the MOLECULAR
PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS by
Richard P. Haugland, 6.sup.th Ed., (1996), and its subsequent
7.sup.th edition and 8.sup.th edition updates issued on CD Rom in
November 1999 and May 2001, respectively, the contents of which are
incorporated by reference, and in other published sources. Examples
1-3, 19, 21-22 and 29-30 describe methods for DDAO phosphate use
and detection.
[0043] The fluorogenic tyramide substrates are combined with the
horseradish peroxidase-labeled, specific binding pair-labeled
target-complex under conditions suitable for the formation of an
immobilized product. The use of labeled tyramides is described in
U.S. Pat. Nos. 5,196,306 and 5,583,001, the contents of which are
incorporated by reference, and in other published sources. Examples
15 describes a method for use and detection of fluorgenic tyramide
substrates.
[0044] In another embodiment of the method, the specific binding
pair member is covalently labeled with a fluorescent dye. The
specific binding pair member selectively binds to its complementary
member/target, to form a fluorescent dye-specific binding-pair
target-complex. The fluorescent dye is detectably distinct from the
dipyrrometheneboron difluoride dye used to counterstain the
immobilized poly(amino acids) but may be a dipyrrometheneboron
difluoride dye of a different chemical structure. Preferably,
however, the fluorescent dye is a xanthene, including fluorescein-
and rhodamine-based dyes or is a carbocyanine, including Cy3 and
Cy5 dyes.
[0045] In one aspect of the invention, stained solid supports are
used to analyze the composition of complex sample mixtures and
additionally to determine the relative amount of a particular
poly(amino acid) in such mixtures. Stained solid supports are also
used to estimate the purity of isolated poly(amino acids) and to
determine the degree of proteolytic degradation of the immobilized
poly(amino acids). In addition, electrophoretic mobility is
optionally used to provide a measure of the molecular weight of
uncharacterized poly(amino acids) and to analyze subunit
composition for multi-subunit poly(amino acids), as well as to
determine the stoichiometry for the subunits bound in such
proteins. In the case of isoelectric focusing electrophoresis
(IEF), electrophoretic mobility is used to provide a measure of the
net molecular charge possessed by the poly(amino acid).
[0046] The two-dimensional electrophoresis portion of the method of
this invention can be performed according to known procedures,
which may vary widely. In a typical procedure, the sample is first
given a linear separation in an elongate or rod-shaped gel, with an
electric potential imposed along the length of the gel. Migration
and separation thus occur along the gel axis until the proteins in
the sample are distributed among zones positioned along the length
of the gel. This is followed by placement of the elongate gel along
one edge of a slab gel, and the imposition of an electric potential
in a direction lying within the plane of the slab gel and
perpendicular to the edge where the elongate gel is placed. The
proteins from each zone of the elongate gel migrate into the slab
gel in the direction transverse to the axis of the elongate gel.
The result is a two-dimensional array of protein spots in the slab
gel. By using one mode of separation or one set of separation
conditions in the first dimension (the elongate gel) and a
different mode or set of separation conditions in the second
dimension (the slab gel), highly effective separations can be
obtained. For example, the first mode may be one based on charge,
such as isoelectric focusing, and the second may be based on
molecular weight.
[0047] Alternatively, the two dimensions of the separation may be
based on the same parameter but may differ in the compositions of
the two gels. The two gels may differ in gel concentration or
chemical components. As a further alternative, the two dimensions
of the separation may be based on the same parameter and performed
in gels of the same composition and concentration, but differ in a
separation condition, such as a stepwise difference in pH, for
example. Still further alternatives are the use of a homogeneous
gel in one of the two dimensions and a gradient gel in the other,
the use of two different protein solubilizers in the two
dimensions, or two different concentrations of the same protein
solubilizer, and the use of a nonchanging buffer system in one
dimension and a changing (gradient, for example) buffer system in
another dimension.
[0048] Transfer of poly(amino acids) from a gel onto a solid
surfaces like a membranes can be carried out using several methods
such as a vacuum, capillary action or by means of an electric field
(electroblotting).
[0049] Poly(amino acids) may be obtained from various sources,
including biological fermentation media and automated protein
synthesizers, as well as prokaryotic cells, eukaryotic cells, virus
particles, tissues, and biological fluids. Suitable biological
fluids include, but are not limited to, urine, cerebrospinal fluid,
blood, lymph fluids, interstitial fluid, cell extracts, mucus,
saliva, sputum, stool, physiological or cell secretions or other
similar fluids. In one embodiment, the poly(amino acids) comprise
the proteome of an animal cell, typically a mammalian cell.
[0050] One aspect of the invention comprises separation of the
poly(amino acids) from the electrophoretic matrix. Another aspect
of the invention further comprises ionization of the poly(amino
acids) and their characterization by mass spectroscopy, or transfer
and subsequent analysis of the poly(amino acids) by Edman
sequencing.
[0051] A further embodiment of the invention is a method of
detecting poly(amino acids) comprising the steps of:
[0052] a. combining a poly(amino acids) immobilized on a solid
support with a labeling mixture that comprises one or more
dipyrrometheneboron difluoride dyes and
[0053] b. incubating a labeling mixture for a sufficient time to
form a covalent bond between the dipyrrometheneboron difluoride
dyes and the immobilized poly(amino acids) to form a dye poly(amino
acid) complex;
[0054] c. removing any unbound dipyrrometheneboron difluoride
dyes;
[0055] d. illuminating the dye-poly(amino acid) complex to yield a
detectable optical response;
[0056] e. using the detectable optical response to detect the
corresponding dye-poly(amino acid) complex.
[0057] In one aspect of this method, the poly(amino acids) are
immobilized on a membrane. The immobilized poly(amino acids) are
typically on a membrane or in an array. In another aspect, the
poly(amino acids) are selectively bound to a corresponding aptamer,
which aptamers are immobilized.
[0058] In the subject arrays and aptamers, the poly(amino acids)
are immobilized to the surface of a solid support. By immobilized
is meant that the poly(amino acids) maintain their position
relative to the rigid support under protein-aptamer complex
formation and washing conditions. As such, the poly(amino acids)
can be non-covalently or covalently associated with the rigid
support surface. Examples of non-covalent association include
non-specific adsorption, specific binding through a specific
binding pair member covalently attached to the support surface, and
entrapment in a matrix material, e.g., a hydrated or dried
separation medium. Examples of covalent binding include covalent
bonds formed between the poly(amino acid) and a functional group
present on the surface of the rigid support, e.g., OH, where the
functional group may be naturally occurring or present as a member
of an introduced linking group, as described in greater detail
below.
[0059] By solid is meant that the support is rigid and does not
readily bend, i.e. the support is not flexible. Examples of solid
materials that are not rigid supports with respect to the present
invention include membranes, flexible plastic films, and the like.
As such, the solid support of the subject arrays and aptamers are
sufficient to provide physical support and structure to the
polymeric targets present thereon under the assay conditions in
which the array is employed, particularly under high-throughput
handling conditions.
[0060] The solid support upon which the subject patterns of
poly(amino acids) are presented in the subject arrays or aptamers
may take a variety of configurations, ranging from simple to
complex, depending on the intended use of the array. Thus, the
support could have an overall slide or plate configuration, such as
a rectangular or disc configuration, where an overall rectangular
configuration, as found in standard microplates and microscope
slides, is preferred. Generally, the length of the rigid supports
will be at least about 1 cm and may be as great as 40 cm or more,
but will usually not exceed about 30 cm and may often not exceed
about 15 cm. The width of the rigid support will generally be at
least about 1 cm and may be as great as 30 cm, but will usually not
exceed 20 cm and will often not exceed 10 cm. The height of the
solid support will generally range from 0.01 mm to 10 mm, depending
at least in part on the material from which the solid support is
fabricated and the thickness of the material required to provide
the requisite rigidity.
[0061] The solid support of the subject arrays or aptamers may be
fabricated from a variety of materials. The materials from which
the substrate is fabricated should ideally exhibit a low level of
non-specific binding of specific binding pair members during
protein-aptamer complex formation or specific binding events. In
many situations, it will also be preferable to employ a material
that is transparent to visible and/or UV light. Specific materials
of interest include: glass; plastics, e.g.,
polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate,
and blends thereof, and the like; metals, e.g., gold, platinum, and
the like; etc.
[0062] The solid support of the subject arrays or aptamers comprise
at least one surface on which the array or aptamers are present,
where the surface may be smooth or substantially planar, or have
irregularities, such as depressions or elevations. The surface may
be modified with one or more different layers of compounds that
serve to modulate the properties of the surface in a desirable
manner. Such modification layers, when present, will generally
range in thickness from a monomolecular thickness to about 1 mm,
usually from a monomolecular thickness to about 0.1 mm and more
usually from a monomolecular thickness to about 0.001 mm.
Modification layers of interest include: inorganic and organic
layers such as metals, metal oxides, polymers, small organic
molecules and the like. Polymeric layers of interest include layers
of: peptides, proteins, poly(nucleic acids) or mimetics thereof,
e.g., peptide nucleic acids and the like; polysaccharides,
phospholipids, polyurethanes, polyesters, polycarbonates,
polyureas, polyamides, polyethyleneimines, polyarylene sulfides,
polysiloxanes, polyimides, polyacetates, and the like, where the
polymers may be hetero- or homopolymeric, and may or may not have
separate functional moieties attached thereto, e.g.,
conjugated.
[0063] Protein microarray production is a highly automated process,
using either pin-based or microdispensing liquid handling robots to
arrange biological samples on a flat surface for to generate
high-density protein arrays and microarrays. This method involves
using gridding robots that transfer either DNA or the corresponding
protein expressed, from microplates on to poly(vinylidene
difluoride) (Hybond-PVDF, Amersham) membranes in high-density
grids. Protein microarrays can also be generated by spotting the
purified protein from liquid expression cultures using a transfer
stamp mounted onto a flat-bed spotting robot. In addition, there
have been many other developments in this area, which have been
recently reviewed. Such developments include the generation of
low-density protein arrays on filter membranes, such as the
universal protein array system (UPA). This is based on the 96-well
microplate format. In this system, protein microarrays have been
printed on an optically flat glass plate containing 96 wells formed
by an enclosing hydrophobic Teflon mask. Inside the wells, arrays
of 144 elements each, were spotted using a 36-capillary-based print
head attached to a precise, high-speed, X-Y-Z robot. Standard ELISA
techniques and a scanning CCD detector were used for imaging of
arrayed antigens. Other approaches to protein microarrays that have
been reported use either photolithography of silane or gold
monolayers, combining microwells with microsphere sensors or
ink-jetting onto polystyrene film. Such formats involve generating
miniaturized immunoassay formats by patterning of single proteins
(e.g., BSA, avidin or monoclonal antibodies). The current invention
will aid in the quality control and detection of proteins
immobilized on the various surfaces.
[0064] An optical response is detected qualitatively, or optionally
quantitatively, by means that include visual inspection, CCD
cameras, video cameras, photographic film, or the use of
instrumentation such as laser scanning devices, fluorometers,
photodiodes, quantum counters, epifluorescence microscopes,
scanning microscopes, flow cytometers, fluorescence microplate
readers, or by means for amplifying the signal such as
photomultiplier tubes. Recording the optical response using
POLAROID film results in enhanced sensitivity of signal. Responses
to other detectable labels (radioactivity, electron spin, magnetic
properties etc) is determined with instrumentation appropriate to
the label.
Applications
[0065] The instant invention has useful applications in basic
proteomic research applications, including but not limited to, 2-D
gels in combination with Western blotting, aptamer technology,
high-throughput screening, microarray technology, drug development,
and medical diagnostics. The methods and kits of the invention can
be used in a variety of assay formats for diagnostic applications
in the disciplines of microbiology, immunology, hematology and
blood transfusion. In general, the methods and kits of the current
invention provide a versatile and convenient means to enhance the
sensitivity and quantitative aspects of any assay that uses
immobilized poly(amino acids) as part of its methodology.
[0066] The combination of Western blotting and high-resolution 2-D
gel electrophoresis represents a rapid and simple method toward the
identification of protein spots in complex mixtures, such as
lysates from organs, tissues, cells, and body fluids. Frequently
the 2-D gel electrophoresed proteins are transferred onto a solid
support (blotting). These "blotted proteins" are analyzed for their
antigenic properties (immunoblotting) using antibodies, though
other protein-selective probes such as DNA, RNA, labeled aptamers,
protein-binding ligands such as fluorescent penicillin analogs for
penicillin-binding proteins and lectins can be used. To obtain a
reference point in order to identity the protein of interest, a
total protein pattern from the 2-D gel is required.
[0067] The instant invention can be used in conjunction with
methods used to study poly(amino acids) as a diagnostic or
prognostic using aptamers. Aptamers are DNA or RNA molecules that
bind specific proteins. The specificity of aptamers allows them to
distinguish between even closely related proteins, a key advantage
for large-scale comprehensive diagnostics and for research
proteomics. In addition to the superior specificity and affinity of
aptamers, their advantages over current capture agent technologies
include direct detection and cost efficiency. The use of aptamers
allows screening of bodily fluids. For example, arrays of aptamers
are bound to a solid support, and a sample is applied. The sample
can be any biological sample including but not limited to blood or
urine. Unbound protein is washed off, and the instant inventions is
used to label that label proteins that are bound to the aptamer but
not nucleic acids used to generate protein profiles. The instant
invention can distinguish functional groups of amino acids from
those of nucleic acids and will give a direct readout of proteins
on the solid support, even in the presence of nucleic acids, which
are essentially unreactive to the preferred amine-reactive dyes of
this invention. Conversely, labeled aptamers can be used as
protein-selective detection reagents for immobilized proteins.
[0068] The major advantages of using aptamers in high-throughput
screening assays are speed of aptamer identification, high affinity
of aptamers for protein targets, relatively large aptamer-protein
interaction surfaces, and compatibility with various
labeling/detection strategies. Aptamers may be particularly useful
in high-throughput screening assays with protein targets that have
no known binding partners such as orphan receptors. Since aptamers
that bind to proteins are often specific and potent antagonists of
protein function, the use of aptamers for target validation can be
coupled with their subsequent use in high-throughput screening.
Given the large size of many conventional and combinatorial
libraries and the rapid increase in the number of possible
therapeutic targets, the speed with which efficient high-throughput
screening assays can be developed can be a rate-limiting step in
the discovery process. Applications of the methods and kits of the
current invention for detection of protein binding to immobilized
aptamer arrays are expected to have the same advantages that they
do for detection of proteins immobilized on blots since the same
light sources are used in array readers as in commercially
available blot readers.
[0069] Proteomics will also play an important role for drug
discovery and development. Proteomics is the link between genes,
proteins and disease. Many of the best-selling drugs either act by
targeting proteins or are proteins. In addition, many molecular
markers of disease, the basis of diagnostics, are proteins whose
patterns of expression can be used as a guide to drug design.
Application of proteomics to study underlying pharmaceutical
mechanisms and to use this information for drug development is
referred to as pharmaceutical proteomics. Unlike classical genomic
approaches that discover genes related to a disease, proteomics
could characterize the disease process directly by finding sets of
proteins (pathways or clusters) that participate together in
causing the disease. The same technology is used to study the
effects of candidate drugs intended to reverse a disease
process.
EXAMPLES
[0070] The following examples describe specific aspects of the
invention to illustrate the invention and to provide a description
of the methods for those of skill in the art. The examples should
not be construed as limiting the invention, as the examples merely
provide specific methodology useful in understanding and practicing
the invention. The reagents employed in the examples are
commercially available or can be prepared using commercially
available instrumentation, methods, or reagents known in the art.
The foregoing examples illustrate various aspects of the invention
and practice of the methods of the invention. Each of the
references cited in the examples is incorporated herein by
reference in its entirety. The examples are not intended to provide
an exhaustive description of the many different embodiments of the
invention nor to limit the selection of suitable dyes beyond what
has already been described above. Thus, although the forgoing
invention has been described in some detail by way of illustration
and example for purposes of clarity of understanding, those of
ordinary skill in the art will realize readily that many changes
and modifications can be made thereto without departing from the
spirit or scope of the appended claims.
1. Simultaneous Dichromatic Detection of Total Protein and a
Specific Target Protein Using BODIPY FL-X Succinimidyl Ester and
DDAO Phosphate.
[0071] A mixture of a dilution series of tubulin (500 ng/lane-0.244
ng/lane) and a constant 250 ng/lane broad range markers (including
myosin, .beta.-galactosidase, phosphorylase b, serum albumin,
ovalbumin, carbonic anhydrase, trypsin inhibitor, lysozyme and
aprotinin) were separated by SDS-polyacrylamide gel electrophoresis
utilizing a 4% T, 2.6% C stacking gel, pH 6.8 and a 13% T, 2.6% C
separating gel, pH 8.8 according to standard procedures. % T is the
total monomer concentration (acrylamide+crosslinker) expressed in
grams per 100 mL and % C is the percentage crosslinker (e.g.,
N,N'-methylene-bis-acrylamide, N,N'-diacryloylpiperazine or other
suitable agent). After electrophoresis, proteins were transferred
to a poly(vinylidene difluoride) (PVDF) membrane by
electroblotting. The membranes were allowed to dry to minimize loss
of proteins during subsequent manipulations. Membranes were
equilibrated by incubation for 10 minutes (two times) in 10 mM
sodium borate buffer, pH 9.5. After equilibration, the membranes
were stained for 30 minutes with 10 .quadrature.M BODIPY FL-X,
succinimidyl ester in the same buffer. After two 5-10 minute washes
in 100% methanol, the membranes were rinsed with dH.sub.2O and
allowed to air dry. Immunodetection was performed by standard
procedures, including a 1 hour blocking step, 2 hour primary
antibody incubation, and 1 hour alkaline phosphatase-conjugated
secondary antibody incubation at room temperature with agitation.
The blocking buffer contained 0.25% MOWIOL 4-88, 0.5% BSA and 0.2%
Tween 20. To detect the presence of the alkaline
phosphatase-conjugated secondary antibody on the blot, the
membranes were incubated for 30 minutes with 1.25 .mu.g/mL DDAO
phosphate in a buffer containing 1 mM MgCl.sub.2 and 10 mM Tris, pH
9.5. Proteins were viewed using a 300 nm UV epi-illuminator. The
total protein profile appeared green fluorescent while the specific
target was stained red fluorescent. Membranes can also be imaged
using a laser system such as the Fuji FLA-3000 imager utilizing the
633 nm excitation filter and 675 nm emission filter for the DDAO
dye and a 473 nm excitation filter and 520 nm emission filter for
the BODIPY FL-X dye. Other BODIPY dyes can be utilized
similarly.
2. Serial Dichromatic Detection of a Specific Target Using DDAO
Phosphate Followed by Total Protein Detection Using BODIPY FL-X
Succinimidyl Ester.
[0072] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted by standard methods. The
membranes were allowed to dry to minimize loss of proteins during
subsequent manipulations. Immunodetection was performed by standard
procedures. To detect the presence of the alkaline
phosphatase-conjugated secondary antibody on the blot the membranes
were incubated for 30 minutes with 1.25 .mu.g/mL DDAO phosphate in
a buffer containing 1 mM MgCl.sub.2 and 10 mM Tris, pH 9.5. Viewing
membranes by UV illumination or using a 633 nm helium-neon laser
scanner revealed a red-fluorescent signal associated with the
target protein. The membranes were allowed to dry and were then
equilibrated by incubation for 10 minutes (two times) in 10 mM
sodium borate buffer, pH 9.5. After equilibration, the membranes
were stained for 30 minutes with 10 .quadrature.M BODIPY FL-X,
succinimidyl ester in the same buffer. After 5-10 minute washes in
100% methanol the membranes were rinsed with dH.sub.2O and allowed
to dry. Proteins were viewed using a 300 nm UV epi-illuminator. The
total protein profile appeared green fluorescent while the specific
target was not detected because the DDAO dye does not precipitate
on the membrane and was washed off during subsequent staining
steps. Other BODIPY dyes can be utilized similarly.
3. Stripping Dichromatic Membranes of the DDAO Signal to Allow
Re-Probing with Another Antibody.
[0073] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and subsequently electroblotted by standard
methods, as described in the previous examples. The membranes were
allowed to dry to minimize loss of proteins during subsequent
manipulations. Membranes were equilibrated by incubation for 10
minutes (two times) in 10 mM sodium borate buffer, pH 9.5. After
equilibration, the membranes were stained for 30 minutes with 10
.quadrature.M BODIPY FL-X, SE in the same buffer. After 5-10 minute
washes in 100% methanol, the membranes were rinsed with dH.sub.2O
and allowed to dry. Immunodetection was performed by standard
methods, as described in the previous examples. To detect the
presence of the alkaline phosphatase-conjugated secondary antibody
on the blot the membranes were incubated for 30 minutes with 1.25
.mu.g/mL DDAO phosphate (Molecular Probes, Inc, Eugene, Oreg.) in a
buffer containing 1 mM MgCl.sub.2 and 10 mM Tris, pH 9.5. Proteins
were viewed using a 300 nm UV epi-illuminator. The total protein
profile appeared green fluorescent while the specific target was
stained red fluorescent. After initial verification of protein
labeling, the membranes were stripped by incubating them for 1
hour, at 500, in buffer containing 2% SDS, 62.5 mM Tris-HCl, pH 6.8
and 50 mM DTT. After rinsing the membranes with 50 mM Tris, pH 7.5
and 150 mM NaCl, proteins previously detected with the DDAO dye
were no longer visible using a 300 nm UV epi-illuminator. The total
protein profile still appeared green fluorescent. These membranes
could be incubated with another monoclonal antibody and an alkaline
phosphatase- or horseradish peroxidase-conjugated secondary
antibody in combination with suitable enzyme substrates, permitting
detection of a different antigen on the blot. The green-fluorescent
BODIPY FL dye signal remained, even after the stripping and
re-probing steps. Other BODIPY dyes can be utilized similarly.
4. Simultaneous Dichromatic Detection of Total Protein and a
Specific Target Protein Using BODIPY TR-X Succinimidyl Ester and
ELF 97 Phosphate.
[0074] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted by standard methods, as
described in Example 1. The membranes were allowed to dry to
minimize loss of proteins during subsequent manipulations.
Membranes were equilibrated by incubation for 10 minutes (two
times) in 10 mM sodium borate buffer, pH 9.5. After equilibration,
the membranes were stained for 30 minutes with 10 .quadrature.M
BODIPY TR-X, SE in the same buffer. After two 5-10 minute washes in
100% methanol the membranes were rinsed with dH.sub.2O and allowed
to dry. Immunodetection was performed by standard procedures. To
detect the presence of the alkaline phosphatase-conjugated
secondary antibody on the blot the membranes were incubated for 30
minutes with 10 .mu.g/mL ELF 97 phosphate in a buffer containing 1
mM MgCl.sub.2 and 10 mM Tris, pH 9.5. Proteins were viewed using
300 nm UV epi-illumination. The total protein profile appeared red
fluorescent while the specific target was stained green
fluorescent. Labeling with the ELF 97 phosphate substrate prior to
labeling with BODIPY TR-X succinimidyl ester produced the same
results. Other BODIPY dyes can be utilized similarly.
5. Dichromatic Detection Using BODIPY TR-X Succinimidyl Ester and
ELF 97 Phosphate Followed by Stripping of the Membranes for
Re-Probing.
[0075] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted, as described in Example 1. The
membranes were allowed to dry to minimize loss of proteins during
subsequent manipulations. Membranes were equilibrated by incubation
for 10 minutes (two times) in 10 mM sodium borate buffer, pH 9.5.
After equilibration, the membranes were stained for 30 minutes with
10 .quadrature.M BODIPY TR-X, SE in the same buffer. After 5-10
minute washes in 100% methanol the membranes were rinsed with
dH.sub.2O and allowed to dry. Immunodetection was performed by
standard procedures. To detect the presence of the alkaline
phosphatase-conjugated secondary antibody on the blot the membranes
were incubated for 30 minutes with 10 .mu.g/mL ELF 97 phosphate in
a buffer containing 1 mM MgCl.sub.2 and 10 mM Tris, pH 9.5.
Proteins were viewed using 300 nm UV epi-illumination. The total
protein profile appeared red fluorescent while the specific target
was stained green fluorescent. After initial verification of
protein labeling, the membranes were incubated for 1 hour, at
50.degree. C., in buffer containing 2% SDS, 62.5 mM Tris-HCl, pH
6.8 and 50 mM DTT. After rinsing the membranes with 50 mM Tris, pH
7.5, 150 mM NaCl, proteins previously detected with ELF 97
phosphate were still visible using a 300 nm UV epi-illuminator.
This demonstrates that the ELF 97 alcohol precipitate was
permanent. The total protein profile appeared as red-fluorescent
bands. Other BODIPY dyes can be utilized similarly.
6. Simultaneous Dichromatic Detection of a Specific Target Using
BCIP/NBT Followed by Total Protein Detection Using BODIPY TR-X
Succinimidyl Ester.
[0076] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted as described in Example 1. The
membranes were allowed to dry to minimize loss of proteins during
subsequent manipulations. Immunodetection was performed by standard
procedures. To detect the presence of the alkaline
phosphatase-conjugated secondary antibody on the blot the membranes
were incubated for 3-60 minutes in NBT/BCIP in a buffer containing
100 mM Tris, pH 9.5, 50 mM MgCl.sub.2 and 100 mM NaCl. The endpoint
of incubation was determined by eye, based upon color development.
The membranes were allowed to dry and were then equilibrated by
incubation for 10 minutes (two times) in 10 mM sodium borate
buffer, pH 9.5. After equilibration, the membranes were stained for
30 minutes with 10 .quadrature.M BODIPY TR-X, succinimidyl ester in
the same buffer. After two 5-10 minute washes in 100% methanol the
membranes were rinsed with dH.sub.2O and allowed to dry. The total
protein profile was viewed using 300 nm UV epi-illumination and
appeared red fluorescent while the NBT/BCIP signal associated with
the specific target appeared blue colored when using white light to
visualize the signal. NBT/BCIP was normally purple in color but
when followed by BODIPY TR-X, succinimidyl ester it appeared blue.
Other BODIPY dyes can be utilized similarly.
7. Serial Dichromatic Detection of Total Protein Using BODIPY TR-X
Succinimidyl Ester Followed by a Specific Target Using
NBT/BCIP.
[0077] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted, as described in Example 1. The
membranes were allowed to dry to minimize loss of proteins during
subsequent manipulations. Membranes were then equilibrated by
incubation for 10 minutes (two times) in 10 mM sodium borate
buffer, pH 9.5. After equilibration, the membranes were stained for
30 minutes with 10 .quadrature.M BODIPY TR-X, succinimidyl ester in
the same buffer. After two 5-10 minute washes in 100% methanol the
membranes were rinsed with dH.sub.2O and allowed to dry. Proteins
appeared as red-fluorescent bands when visualized by UV
epi-illumination. Immunodetection was performed by standard
procedures. To detect the presence of the alkaline
phosphatase-conjugated secondary antibody on the blot the membranes
were incubated for 3-60 minutes in NBT/BCIP in a buffer containing
100 mM Tris, pH 9.5, 50 mM MgCl.sub.2, and 100 mM NaCl. The
endpoint of incubation was determined by eye based on color
development. The red fluorescence of the BODIPY dye was quenched by
the NBT/BCIP. The NBT/BCIP precipitate appears purple by eye. Other
BODIPY dyes can be utilized similarly.
8. Simultaneous Dichromatic Detection of Total Protein and a
Specific Target Using BODIPY TR-X Succinimidyl Ester and
Fluoroblot.TM. Peroxidase Substrate.
[0078] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted, as described in Example 1. The
membranes were then allowed to dry to minimize loss of proteins
during subsequent manipulations. Membranes were then equilibrated
by incubation for 10 minutes (two times) in 10 mM sodium borate
buffer, pH 9.5. After equilibration, the membranes were stained for
30 minutes with 10 .mu.M BODIPY TR-X, SE in the same buffer. After
5-10 minute washes in 100% methanol the membranes were rinsed with
dH.sub.2O and allowed to dry. Immunoblotting was performed by
standard procedures including a 1 hour blocking step, 2 hour
primary antibody incubation, and 1 hour horseradish
peroxidase-conjugated secondary antibody incubation at room
temperature with agitation. The blocker included 0.25% MOWIOL 4-88,
0.5% BSA, and 0.2% Tween 20. To detect the presence of the
horseradish peroxidase-conjugated secondary antibody on the blot
the membranes were incubated for 30 minutes in Fluoroblot
peroxidase substrate (Pierce Chemical Company, Milwaukee, Wis.)
diluted 1:1 in stable peroxidase buffer. Proteins were viewed using
300 nm UV epi-illumination. The total protein profile appeared red
fluorescent while the specific target was stained green
fluorescent. The Fluoroblot dye-labeled proteins were viewed best
when the membranes were wet because the fluorescence was
considerably reduced upon drying. Other BODIPY dyes can be utilized
similarly.
9. Serial Dichromatic Detection of a Specific Target Followed by
Total Protein Detection Using Fluoroblot Peroxidase Substrate and
BODIPY Dyes.
[0079] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted, as described in Example 1. The
membranes were then allowed to dry to minimize loss of proteins
during subsequent manipulations. Immunodetection was performed by
standard procedures. To detect the presence of the horseradish
peroxidase-conjugated secondary antibody on the blot the membranes
were incubated for 30 minutes in Fluoroblot peroxidase substrate
diluted 1:1 in stable peroxidase buffer. Upon viewing with a 300 nm
epi-illumination source, the target protein appeared green
fluorescent. The membranes were allowed to dry and were then
equilibrated by incubation for 10 minutes (two times) in 10 mM
sodium borate buffer, pH 9.5. After equilibration, the membranes
were stained for 30 minutes with 10 .quadrature.M BODIPY TR-X,
succinimidyl ester in the same buffer. After 5-10 minute washes in
100% methanol the membranes were rinsed with dH.sub.2O and allowed
to dry. Proteins were viewed using 300 nm UV epi-illumination. The
total protein profile appeared fluorescent red while the specific
target was no longer detected because the Fluoroblot dye was washed
off the membrane in the subsequent BODIPY dye staining steps. Other
BODIPY dyes can be utilized similarly.
10. Simultaneous Dichromatic Detection of Total Protein and a
Specific Target Using BODIPY TR-X Succinimidyl Ester and Amplex
Gold Substrate.
[0080] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted, as described in Example 1. The
membranes were then allowed to dry to minimize loss of proteins
during subsequent manipulations. Membranes were then equilibrated
by incubation for 10 minutes (two times) in 10 mM sodium borate
buffer, pH 9.5. After equilibration, the membranes were stained for
30 minutes with 10 .quadrature.M BODIPY TR-X, SE in the same
buffer. After 5-10 minute washes in 100% methanol the membranes
were rinsed with dH.sub.2O and allowed to dry. Immunoblotting was
performed by standard procedures including a 1 hour blocking step,
2 hour primary antibody incubation, and 1 hour horseradish
peroxidase-conjugated secondary antibody incubation at room
temperature with agitation. The blocker included 0.25% MOWIOL 4-88,
0.5% BSA, and 0.2% Tween 20. To detect the presence of the
horseradish peroxidase-conjugated secondary antibody on the blot
the membranes were incubated for 30 minutes with
50.quadrature..quadrature. Amplex Gold in a buffer containing 1 mM
MgCl.sub.2, 280 .mu.M ZnCl.sub.2, 10 mM Tris, pH 9.5 and 200 .mu.M
H.sub.2O.sub.2. Proteins were viewed using a 300 nm UV
epi-illuminator. The total protein profile appeared red fluorescent
while the specific target was labeled goldenrod. Proteins were
viewed using a 300 nm UV epi-illuminator. Membranes could also have
been imaged using a laser system such as the Fuji FLA-3000 imager
utilizing the 633 nm excitation filter and 675 nm emission filter
for the BODIPY TR-X dye and a 532 nm excitation filter and 580 nm
emission filter for Amplex Gold. Other BODIPY dyes can be utilized
similarly.
11. Serial Dichromatic Detection of Total Protein and a Specific
Target Using BODIPY TR-X Succinimidyl Ester and
4-chloro-1-naphthol.
[0081] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted, as described in Example 1. The
membranes were allowed to dry to minimize loss of proteins during
subsequent manipulations. Membranes were then equilibrated by
incubation for 10 minutes (two times) in 10 mM sodium borate
buffer, pH 9.5. After equilibration, the membranes were stained for
30 minutes with 10 .quadrature.M BODIPY TR-X, SE in the same
buffer. After two 5-10 minute washes in 100% methanol the membranes
were rinsed with dH.sub.2O and allowed to dry. Proteins appeared as
red-fluorescent bands upon 300 nm UV epi-illumination.
Immunodetection was performed by standard procedures. To detect the
presence of the horseradish peroxidase-conjugated secondary
antibody on the blot the membranes were incubated for 3-60 minutes
in 4-chloro-1-naphthol solution (0.48 mM 4-chloro-1-naphthol, 50 mM
Tris HCl, 0.2 mM NaCl and 17% methanol) to which was added 0.01%
(v/v) hydrogen peroxide. The endpoint of incubation was determined
by eye based upon color development. The fluorescence of the total
protein profile was quenched by the 4-chloro-1-naphthol. The
4-chloro-1-naphthol labeling of the specific target protein appears
purple by eye when viewed using white-light illumination. Other
BODIPY dyes can be utilized similarly.
12. Simultaneous Dichromatic Detection of a Specific Target Using
4-chloro-1-naphthol Followed by Total Protein Staining Using BODIPY
TR-X Succinimidyl Ester.
[0082] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted as described in Example 1. The
membranes were allowed to dry to minimize loss of proteins during
subsequent manipulations. Immunodetection was performed by standard
procedures. To detect the presence of the horseradish
peroxidase-conjugated secondary antibody on the blot the membranes
were incubated for 3-60 minutes in 4-chloro-1-naphthol solution
(0.48 mM 4-chloro-1-naphthol, 50 mM Tris HCl, 0.2 mM NaCl and 17%
methanol) to which 0.01% (v/v) hydrogen peroxide was added. The
membranes were allowed to dry and were then equilibrated by
incubation for 10 minutes (two times) in 10 mM sodium borate
buffer, pH 9.5. After equilibration, the membranes were stained for
30 minutes with 10 .quadrature.M BODIPY TR-X, SE in the same
buffer. After two 5-10 minute washes in 100% methanol the membranes
were rinsed with dH.sub.2O and allowed to dry. Proteins were viewed
using 300 nm UV epi-illumination. The total protein profile
appeared red fluorescent. The specific target appears purple when
viewed by white-light illumination. Other BODIPY dyes can be
utilized similarly.
13. Serial Dichromatic Detection of Total Protein and a Specific
Target Using BODIPY TR-X Succinimidyl Ester and
3,3'-diaminobenzidine tetrahydrochloride.
[0083] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted as described in Example 1. The
membranes were then allowed to dry to minimize loss of proteins
during subsequent manipulations. Membranes were then equilibrated
by incubation for 10 minutes (two times) in 10 mM sodium borate
buffer, pH 9.5. After equilibration, the membranes were stained for
30 minutes with 10 .mu.M BODIPY TR-X, succinimidyl ester in the
same buffer. After 5-10 minute washes in 100% methanol the
membranes were rinsed with dH.sub.2O and allowed to dry. Upon
viewing with a 300 nm UV epi-illumination source, proteins appeared
as red-fluorescent bands. Immunodetection was performed by standard
procedures. To detect the presence of the horseradish
peroxidase-conjugated secondary antibody on the blot the membranes
were incubated for 3-60 minutes with 0.6 mg/mL
3,3'-diaminobenzidine in 50 mM Tris, pH 7.6, 0.03% hydrogen
peroxide (v/v). The endpoint of incubation was determined by eye
based upon color development. The fluorescence of the total protein
profile was quenched by the 3,3'-diaminobenzidine. The
3,3'-diaminobenzidine labeling the specific target protein appeared
brown on the blot when viewed by white-light illumination. Other
BODIPY dyes can be utilized similarly.
14. Simultaneous Dichromatic Detection of Total Protein and a
Specific Target Using BODIPY TR-X Succinimidyl Ester and
3,3'-diaminobenzidine.
[0084] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted as described in Example 1. The
membranes were then allowed to dry to minimize loss of proteins
during subsequent manipulations. Immunoblotting was performed by
standard procedures. To detect the presence of the horseradish
peroxidase-conjugated secondary antibody on the blot the membranes
were incubated for 3-60 minutes with 0.6 mg/mL
3,3'-diaminobenzidine in 50 mM Tris, pH 7.6, 0.03% hydrogen
peroxide (v/v). The membranes were allowed to dry and were then
equilibrated by incubation for 10 minutes (two times) in 10 mM
sodium borate buffer, pH 9.5. After equilibration, the membranes
were stained for 30 minutes with 10 .mu.M BODIPY TR-X, SE in the
same buffer. After 5-10 minute washes in 100% methanol, the
membranes were rinsed with dH.sub.2O and allowed to dry. Proteins
were viewed as red-fluorescent bands using 300 nm UV
epi-illumination. The specific target was labeled brown and was
easily viewed using white-light illumination. Other BODIPY dyes can
be utilized similarly.
15. Simultaneous Dichromatic Detection of a Specific Target Using
Alexa Fluor 488 Tyramide Conjugate and Total Protein Using BODIPY
TR-X Succinimidyl Ester.
[0085] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted as described in Example 1. The
membranes were then allowed to dry to minimize loss of proteins
during subsequent manipulations. Immunodetection was performed by
standard procedures. To detect the presence of the horseradish
peroxidase-conjugated secondary antibody on the blot the membranes
were incubated for 30 minutes in 5 .mu.M Alexa Fluor 488 tyramide
(Molecular Probes, Inc, Eugene Oreg., U.S. Pat. No. 6,130,101 and
U.S. patent Ser. No. 09/969,853, incorporated by reference) in 50
mM Tris, pH 7.5 and 150 mM NaCl to which was added 0.03% hydrogen
peroxide (v/v). The membranes were allowed to dry and were then
equilibrated by incubation for 10 minutes (two times) in 10 mM
sodium borate buffer, pH 9.5. After equilibration, the membranes
were stained for 30 minutes with 10 .quadrature.M BODIPY TR-X, SE
in the same buffer. After 5-10 minute washes in 100% methanol the
membranes were rinsed with dH.sub.2O and allowed to dry. Proteins
can be viewed using 300 nm UV epi-illumination. The total protein
profile appeared red fluorescent while the specific target appeared
green fluorescent. Other BODIPY dyes can be utilized similarly in
combination with appropriately selected fluorescent tyramide
derivatives.
16. Simultaneous Dichromatic Detection of Glycoproteins and Total
Proteins Using BODIPY TR-X Succinimidyl Ester and PRO-Q EMERALD 300
Glycoprotein Stain on Membranes.
[0086] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted, as described in Example 1. The
membranes were then allowed to dry to minimize loss of proteins
during subsequent manipulations. The membranes were then
equilibrated by incubation for 10 minutes (two times) in 10 mM
sodium borate buffer, pH 9.5. After equilibration, the membranes
were stained for 30 minutes with 10 .mu.M BODIPY TR-X, SE in the
same buffer. After 5-10 minute washes in 100% methanol the
membranes were rinsed with dH.sub.2O and allowed to dry. The
membranes were then fixed in 50% methanol, oxidized in 1% periodic
acid in 3% acetic acid and stained with 5 .quadrature.M PRO-Q
EMERALD 300 in 2% DMF, 250 mM MgCl.sub.2, and 3% acetic acid. After
staining, the membranes were rinsed in 3% acetic acid followed by
dH.sub.2O and allowed to dry. Proteins were viewed using 300 nm UV
epi-illumination. The total protein profile appeared red
fluorescent while the glycoproteins were green fluorescent.
Labeling with the PRO-Q EMERALD 300 dye prior to labeling with
BODIPY TR-X succinimidyl ester produced the same results. Other
BODIPY dyes can be utilized similarly.
17. Simultaneous Dichromatic Detection of Glycoproteins and Total
Proteins Using BODIPY TR-X Succinimidyl Ester and PRO-Q EMERALD 300
Glycoprotein Stain in Gels.
[0087] Proteins were separated by SDS-polyacrylamide gel
electrophoresis, as described in Example 1. After electrophoresis,
gels were fixed overnight in MeOH:acetic acid:H.sub.2O (5:1:1).
They were then washed and equilibrated in 10 mM sodium borate
buffer, pH 9.5, and stained in 10 .mu.M BODIPY TR-X, SE in the same
buffer. After washing, the gels were fixed in 50% methanol,
oxidized in 1% periodic acid in 3% acetic acid and stained with 5
.quadrature.M PRO-Q EMERALD 300 in 2% DMF, 250 mM MgCl.sub.2, and
3% acetic acid. After staining, the gels were rinsed in 3% acetic
acid followed by dH.sub.2O. Proteins stained with PRO-Q EMERALD 300
can be viewed using an imaging system such as the Roche Lumilmager,
which utilizes a UV light box and a CCD camera. PRO-Q EMERALD 300
dye-stained proteins can be visualized using the 520 nm+/-20
emission filter. BODIPY TR-X dye-conjugated proteins were
visualized and separated from PRO-Q EMERALD 300 dye-stained
proteins using the 590/10 excitation filter and a 625/30 emission
filter.
18. Simultaneous Dichromatic Detection of Glycoproteins Using
Lectin Concanavalin A, Alkaline Phosphatase Conjugate and BODIPY
TR-X Succinimidyl Ester.
[0088] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted, as described in Example 1. The
membranes were then allowed to dry to minimize loss of proteins
during subsequent manipulations. The membranes were then
equilibrated by incubation for 10 minutes (two times) in 10 mM
sodium borate buffer, pH 9.5. After equilibration, the membranes
were stained for 30 minutes with 10 .mu.M BODIPY TR-X, SE in the
same buffer. After two 5-10 minute washes in 100% methanol the
membranes were rinsed with dH.sub.2O and allowed to dry. For
detection of the glycoproteins described above, the membranes were
blocked then incubated in 1 .quadrature.g/mL concanavalin
A-alkaline phosphatase in a buffer containing 150 mM mTBS, 50 Tris,
pH 7.5, 0.2% Tween-207, 0.25% Mowiol 4-88, 0.5 mM MgCl.sub.2, and 1
mM CaCl.sub.2. For detection of the concanavalin A-AP conjugate,
the membranes were incubated with 10 .quadrature.g/mL ELF 97
phosphate in a buffer containing 1 mM MgCl.sub.2 and 10 mM Tris, pH
9.5. Proteins were viewed using 300 nm UV epi-illumination.
Concanavalin A binds to glycoproteins containing
.quadrature.-mannosyl and .quadrature.-glucopyranosyl residues. The
total protein profile appeared red fluorescent while the targeted
glycoproteins were stained green fluorescent.
19. Simultaneous Dichromatic Detection of Glycoproteins Using
Lectin Concanavalin A, Alkaline Phosphatase Conjugate and BODIPY
FL-X Succinimidyl Ester.
[0089] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted as described in Example 1. The
membranes were then allowed to dry to minimize loss of proteins
during subsequent manipulations. The membranes were then
equilibrated by incubation for 10 minutes (two times) in 10 mM
sodium borate buffer, pH 9.5. After equilibration, the membranes
were stained for 30 minutes in 10 .quadrature.M BODIPY FL-X, SE in
the same buffer. After two 5-10 minute washes in 100% methanol the
membranes were rinsed with dH.sub.2O and allowed to dry. For
detection of the glycoproteins described above, the membranes were
blocked then incubated in 1 .quadrature.g/mL concanavalin
A-alkaline phosphatase in a buffer containing 150 mM mTBS, 50 Tris,
pH 7.5, 0.2% Tween-20, 0.25% Mowiol 4-88, 0.5 mM MgCl.sub.2 and 1
mM CaCl.sub.2. For detection of the concanavalin A-AP conjugate,
the membranes were incubated in 1.25 .mu.g/mL DDAO phosphate in a
buffer containing 1 mM MgCl.sub.2 and 10 mM Tris, pH 9.5. Proteins
were viewed using a 300 nm UV epi-illuminator. The total protein
profile appeared green fluorescent while the targeted glycoproteins
were stained red fluorescent. Membranes can also be imaged using a
laser system such as the Fuji FLA-3000 imager utilizing the 633 nm
excitation filter and 675 nm emission filter for the DDAO dye and a
473 nm excitation filter and 520 nm emission filter for the BODIPY
FL-X dye.
20. Simultaneous Dichromatic Detection of Glycoproteins Using
Lectin Wheat Germ Agglutinin and BODIPY TR-X Succinimidyl
Ester.
[0090] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted, as described in Example 1. The
membranes were then allowed to dry to minimize loss of proteins
during subsequent manipulations. The membranes were then
equilibrated by incubation for 10 minutes (two times) in 10 mM
sodium borate buffer, pH 9.5. After equilibration, the membranes
were stained for 30 minutes with 10 .mu.M BODIPY TR-X, SE in the
same buffer. After two 5-10 mL washes in 100% methanol the
membranes were rinsed with dH.sub.2O and allowed to dry. For
detection of the glycoproteins described above, the membranes were
blocked then incubated with 0.5 .mu.g/mL wheat germ
agglutinin-alkaline phosphatase in a buffer containing 150 mM mTBS,
50 Tris, pH 7.5, 0.2% Tween-20, 0.25% Mowiol 4-88, 0.5 mM
MgCl.sub.2, and 1 mM CaCl.sub.2. For detection of the wheat germ
agglutinin-AP conjugate, the membranes were incubated with 10
.mu.g/mL ELF 97 phosphate in a buffer containing 1 mM MgCl.sub.2
and 10 mM Tris, pH 9.5. Proteins were viewed using 300 nm UV
epi-illumination. Wheat germ agglutinin binds glycoproteins
containing N-acetylglucosamine and N-acetylneuraminic acid
residues. The total protein profile appeared red fluorescent while
the targeted glycoproteins were stained green fluorescent.
21. Simultaneous Dichromatic Detection of Glycoproteins Using
Lectin Wheat Germ Agglutinin and BODIPY FL-X Succinimidyl
Ester.
[0091] Proteins were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted, as described in Example 1. The
membranes were then allowed to dry to minimize loss of proteins
during subsequent manipulations. The membranes were then
equilibrated by incubation for 10 minutes (two times) in 10 mM
sodium borate buffer, pH 9.5. After equilibration, the membranes
were stained for 30 minutes with 10 .quadrature.M BODIPY FL-X, SE
in the same buffer. After two 5-10 minute washes in 100% methanol
the membranes were rinsed with dH.sub.2O and allowed to dry. For
detection of the glycoproteins described above, the membranes were
blocked then incubated with 0.5 .mu.g/mL wheat germ
agglutinin-alkaline phosphatase in a buffer containing 150 mM mTBS,
50 Tris, pH 7.5, 0.2% Tween-20, 0.25% Mowiol 4-88, 0.5 mM
MgCl.sub.2, and 1 mM CaCl.sub.2. For detection of the wheat germ
agglutinin-AP conjugate, the membranes were incubated with 1.25
.mu.g/ml DDAO phosphate in a buffer containing 1 mM MgCl.sub.2 and
10 mM Tris, pH 9.5. Proteins can be viewed using a 300 nm UV
epi-illuminator. The total protein profile appeared green
fluorescent while the glycoproteins were stained red fluorescent.
Membranes can also be imaged using a laser system such as the Fuji
FLA-3000 imager utilizing the 633 nm excitation filter and 675 nm
emission filter for the DDAO dye and a 473 nm excitation filter and
520 nm emission filter for the BODIPY FL-X dye.
22. Simultaneous Dichromatic Detection of Total Protein and a
Specific Target Protein Using BODIPY FL-X Succinimidyl Ester in 2-D
Electrophoresis and DDAO Phosphate.
[0092] 150 .quadrature.g of a rat fibroblast lysate was applied to
1 mm diameter, 20 cm long isoelectric focusing gels consisting of a
4% T, 2.6% C polyacrylamide gel matrix, containing 9 M urea, 2%
Triton X-100, and 2% carrier ampholytes. Gels were run vertically
for 18,000 volt-hours using 10 mM phosphoric acid and 100 mM sodium
hydroxide as the anode and cathode buffer, respectively.
Isoelectric focusing gels were incubated in 0.3 M Tris base, 0.075
M Tris-HCl, 3% SDS, 0.01% bromophenol blue for two minutes.
Isoelectric focusing gels were then laid on top of 1 mm thick, 20
cm.times.20 cm, 12.5% T, 2.6% C polyacrylamide gels containing 375
mM Tris-base, pH 8.8 and SDS-polyacrylamide gel electrophoresis was
performed according to standard procedures except that the cathode
electrode buffer was 50 mM Tris, 384 mM glycine, 4% sodium dodecyl
sulfate, pH 8.8 while the anode electrode buffer is 25 mM Tris, 192
mM glycine, 2% sodium dodecyl sulfate, pH 8.8. After the second
dimension electrophoresis, proteins were transferred to a 20
cm.times.20 cm piece of poly(vinylidene difluoride) (PVDF) membrane
by electroblotting. The membranes were allowed to dry to minimize
loss of proteins during subsequent manipulations. Membranes were
equilibrated by incubation for 10 minutes (two times) in 10 mM
sodium borate buffer, pH 9.5. After equilibration, the membranes
were stained for 30 minutes with 10 .mu.M BODIPY FL-X, succinimidyl
ester in the same buffer. After two 5-10 minute washes in 100%
methanol, the membranes were rinsed with dH.sub.2O and allowed to
air dry. Immunodetection was performed by standard procedures,
including a 1 hour blocking step, 2 hour primary antibody
incubation, and 1 hour alkaline phosphatase-conjugated secondary
antibody incubation at room temperature with agitation. The
blocking buffer contained 0.25% Mowiol 4-88, 0.5% BSA and 0.2%
Tween 20. To detect the presence of the alkaline
phosphatase-conjugated secondary antibody on the membrane, the
membranes were incubated for 30 minutes with 1.25 .mu.g/mL DDAO
phosphate in a buffer that contained 1 mM MgCl.sub.2 and 10 mM
Tris, pH 9.5. Proteins were viewed using a 300 nm UV
epi-illuminator. The total protein profile appeared green
fluorescent while the specific target was stained red fluorescent.
Membranes can also be imaged using a laser system such as the Fuji
FLA-3000 imager utilizing the 633 nm excitation filter and 675 nm
emission filter for the DDAO dye and a 473 nm excitation filter and
520 nm emission filter for the BODIPY FL-X dye. Other BODIPY dyes
can be utilized similarly.
23. Trichromatic Detection of Two Specific Targets Using ELF 39
Phosphate, Alexa Fluor 350 and BODIPY TR-X Succinimidyl Ester in
2-D Electrophoresis.
[0093] 300 .quadrature.g of bovine heart mitochondria were diluted
to 5 mg/mL into 25 mM Tris, 2 mM EDTA, pH 7.5. Just prior to
extraction, 1 mM PMSF was added, followed by 1%
n-dodecyl-.beta.-D-maltoside for the actual extraction. The
mitochondria were incubated in the detergent for 20 minutes before
the insoluble material was pelleted by centrifugation (10 min,
12,000 rpm). The protein in the supernatant was precipitated with
10% TCA and pelleted by centrifugation. The pellet was resuspended
in urea sample buffer (7 M urea, 2 M thiourea, 1% Zwittergent 3-10,
2% CHAPS, 0.8% carrier Ampholytes) and frozen at -20.degree. C. IPG
strips (3-10 NL) were rehydrated overnight at room temperature in
the same urea buffer (450 mL). The IPG strip was placed into the
pHaser isoelectric focusing system and the sample was applied on a
piece of filter paper at the anodic end. The sample was subjected
to isolelectric focusing at 20.degree. C. for 24.5 h (70,000 Vh)
and then separated on a 20 cm.times.20 cm.times.1 mm second
dimension SDS-polyacrylamide gel (12.5% T, 2.6% C).
[0094] After second dimension electrophoresis, proteins were
transferred to a 20 cm.times.20 cm piece of PVDF membrane by
electroblotting. The membranes were allowed to dry to minimize loss
of proteins during subsequent manipulations. Membranes are
equilibrated by incubation for 10 minutes (two times) in 10 mM
sodium borate buffer, pH 9.5. After equilibration, the membranes
were stained for 30 minutes with 10 .quadrature.M BODIPY TR-X,
succinimidyl ester in the same buffer. After two 5-10 minute washes
in 100% methanol, the membranes were rinsed with dH.sub.2O and
allowed to air dry. Immunodetection was performed by standard
procedures except that two primary antibodies to two different
targets were applied to the membrane. One primary antibody was
directly conjugated to the Alexa Fluor 350 dye and the other was a
mouse monoclonal antibody that was subsequently detected with a
goat anti-mouse-alkaline phosphatase secondary antibody. To detect
the presence of the alkaline phosphatase-conjugated secondary
antibody on the blot, the membranes were incubated for 30 minutes
in 10 .mu.g/mL ELF 97 phosphate in a buffer containing 1 mM
MgCl.sub.2 and 10 mM Tris, pH 9.5. Proteins were viewed using 300
nm UV epi-illumination. The total protein profile appeared red
fluorescent while one specific target was stained green fluorescent
and the other was labeled blue fluorescent.
24. Trichromatic Detection of Two Specific Targets Using ELF 97
Phosphate, Streptavidin Alexa Fluor 350 dye, and BODIPY TR-X
Succinimidyl Ester.
[0095] A mixture of a dilution series of tubulin (500 ng/lane-0.244
ng/lane), biotin-labeled concanavalin-A (500 ng/lane-0.244 ng/lane)
and a constant 250 ng/lane broad range markers (including myosin,
.beta.-galactosidase, phosphorylase b, serum albumin, ovalbumin,
carbonic anhydrase, trypsin inhibitor, lysozyme and aprotinin) were
separated by SDS-polyacrylamide gel electrophoresis and
electroblotted as described in Example 1. The membranes were then
allowed to dry to minimize loss of proteins during subsequent
manipulations. The membranes were then equilibrated by incubation
for 10 minutes (two times) in 10 mM sodium borate buffer, pH 9.5.
After equilibration, the membranes were stained for 30 minutes with
10 .mu.M BODIPY TR-X, SE in the same buffer. After two 5-10 mL
washes in 100% methanol, the membranes were rinsed with dH.sub.2O
and allowed to air dry. Immunodetection was performed by standard
procedures except that membranes were incubated in a mixture of a
secondary goat anti-mouse IgG-alkaline phosphatase antibody and
streptavidin Alexa Fluor 350. To detect the presence of the
alkaline phosphatase-conjugated secondary antibody on the
membranes, the membranes were incubated for 30 minutes with 10
.mu.g/mL ELF 97 phosphate in a buffer containing 1 mM MgCl.sub.2
and 10 mM Tris, pH 9.5. Proteins were viewed using 300 nm UV
epi-illumination. The total protein profile appeared red
fluorescent while one specific target was stained green fluorescent
and the biotinylated proteins were stained blue fluorescent.
25. Trichromatic Detection of Two Specific Targets Using a Goat
Anti-Mouse Secondary Antibody Conjugated to Alexa Fluor 350, ELF 97
Phosphate, and BODIPY TR-X Succinimidyl Ester.
[0096] A mixture of proteins (as in Example 24) was separated by
SDS-polyacrylamide gel electrophoresis and electroblotted, as
described in Example 1. The membranes were then allowed to dry to
minimize loss of proteins during subsequent manipulations. The
membranes were then equilibrated by incubation for 10 minutes (two
times) in 10 mM sodium borate buffer, pH 9.5. After equilibration,
the membranes were stained for 30 minutes with 10 .mu.M BODIPY
TR-X, SE in the same buffer. After two 5-10 minute washes in 100%
methanol, the membranes were rinsed with dH.sub.2O and allowed to
air dry. Immunodetection was performed by standard procedures,
except that membranes were incubated in a mixture of a secondary
goat anti-mouse IgG-Alexa Fluor 350 conjugate and
streptavidin-alkaline phosphatase. To detect the presence of the
alkaline phosphatase-conjugated streptavidin on the blot the
membranes were incubated for 30 minutes with 10 .mu.g/mL ELF 97
phosphate (U.S. Pat. No. 5,316,906, incorporated by reference) in a
buffer containing 1 mM MgCl.sub.2 and 10 mM Tris, pH 9.5. Proteins
were viewed using 300 nm UV epi-illumination. The total protein
profile appeared red fluorescent while one specific target was
stained blue fluorescent and the biotinylated proteins were stained
green fluorescent.
26. Simultaneous Two Color Detection with in-Gel Zymography and
Total-Protein Staining Using BODIPY TR-X Succinimidyl Ester.
[0097] A partially purified protein sample containing an enzyme of
interest, such as .beta.-glucuronidase, was diluted and loaded onto
a standard SDS-polyacrylamide gel without boiling the sample prior
to application. After separation, the gel was washed in 50 mM
NaHPO.sub.4 buffer, pH 7.0 (0.1% Triton X-100) and incubated in the
enzymatic substrate ELF 97 .beta.-glucuronide diluted in the same
buffer (without Triton X-100). Gels were then fixed overnight in
methanol:acetic acid:water (5:1:1). After fixing they were washed
in dH.sub.2O, equilibrated in 10 mM sodium borate buffer pH 9.5,
and then incubated with 10 .mu.M BODIPY TR-X, SE in the same
buffer. Proteins were viewed using 300 nm UV trans-illumination or
epi-illumination. The total protein profile appeared red
fluorescent while the specific target enzyme was green
fluorescent.
27. Total Protein Staining Using BODIPY Succinimidyl Esters.
[0098] Membrane staining:Proteins were separated by
SDS-polyacrylamide gel electrophoresis and electroblotted, as
described in Example 1. The membranes were then equilibrated by
incubation for 10 minutes (two times) in 10 mM sodium borate
buffer, pH 9.5. After equilibration, the blots were stained for 30
minutes with 10 .mu.M of any BODIPY succinimidyl ester in the same
buffer. After two 5-10 mL washes in 100% methanol the membranes
were rinsed with dH.sub.2O and allowed to dry. Proteins on
membranes were viewed using UV epi-illumination.
[0099] Gel staining:Proteins were separated by SDS-polyacrylamide
gel electrophoresis, as described in Example 1. The gels were then
fixed overnight in methanol:acetic acid:water (5:1:1). After
washing with dH.sub.2O (3.times.30 minutes), the gels were
equilibrated in 10 mM sodium borate buffer, pH 9.5. Gels were
stained by incubating them 10 .mu.M any BODIPY succinimidyl ester
in the same buffer. Proteins were viewed using UV
trans-illumination.
28. Screening BODIPY Succinimidyl Esters.
[0100] Standard protein molecular weight markers were serially
diluted two-fold to generate a concentration range of 1000 ng-0.5
ng of protein/lane. The proteins were separated by
SDS-polyacrylamide gel electrophoresis and electroblotted, as
described in Example 1. The membranes were equilibrated by
incubation for 10 minutes (two times) in 10 mM sodium borate
buffer, pH 9.5. After equilibration, two membranes each were
stained with one of the BODIPY succinimidyl ester dyes, in Table 2.
The concentration of dye used was 10 .mu.M in 10 mM sodium borate
buffer, pH 9.5. Membranes were stained for 30 minutes with
agitation. The membranes were then rinsed two times, 5 minutes each
time, in the same buffer. All membranes were then washed 3 times,
10 minutes each time, in 100% methanol. After a final rinse in
dH.sub.2O, the membranes were imaged with a constant 3 second
exposure on the Bio-Rad Fluor S Max MultiImager or similar device.
A UV epi-illumination light source and the 520 or 610 long pass
filters were used. Each dye was then imaged at its optimal exposure
time, carefully avoiding signal saturation. Using the Quantity One
software, the trace density for a particular protein band was
determined and the background subtracted. The background-subtracted
trace density was plotted versus a range of protein concentration
and the linear dynamic range of detection was determined.
Sensitivity was established by determining the lowest amount of
protein visible on the membrane. Brightness of the dyes was
compared when imaged with a common exposure time.
TABLE-US-00002 TABLE 2 Comparable Brightness Values of 16 BODIPY
Succinimidyl Esters (SEs) Intensity @ 3 secs of 250 ng band of
protein Substitute Bovine Soybean Com- Oval- Serum Carbonic Trypsin
pound # Abs Em bumin Albumin Anhydrase Inhibitor 630/650-X 625 640
169452.2 40215.84 193469.6 161528.4 650/665-X 646 660 31163.88
16294.89 30194.71 26319.33 TR-X 588 616 436280.4 141786.4 558595.7
533819.3 TMR-X 544 570 382633.5 140534.5 564315.7 541307.9 FL
Br.sub.2 530 545 50639.4 30512.31 70927.13 55972.11 R6G 528 547
116884 54146.32 227844.1 228890.7 FL C.sub.5 504 511 36495.05
36574.32 63640.23 49177.72 R6G-X 529 547 219786.1 148676.7 386058.2
300411.8 FL 502 510 54071.31 65458.51 71486.49 62468.47 530/550 534
551 361990.8 226221 438350.6 180694.7 493/503 500 509 24401.56
25658.76 54132.56 23722.79 558/568 559 568 130769.9 37031.36
253114.8 254121.8 564/570 563 569 383091.9 171803.2 605554.4
563892.1 576/589 575 588 78230.08 65072.49 202045.3 101140.1
581/591 581 591 208951.4 160171.6 319791.2 144085.5 FL-X 504 510
153667.2 181723.1 282973 188718.2
TABLE-US-00003 TABLE 3 Linearity and Sensitivity of Detection
Values of 16 BODIPY Succinimidyl Esters (SEs) and fluorescein
isothiocyanate (FITC). Sensitivity (ng of protein) Bovine Soybean
Linearity Optimal Serum Carbonic Trypsin Determined Compound # Abs
Em Exposure* Albumin Ovalbumin Anhydrase Inhibitor Using CA
630/650-X 625 640 4 secs 7.8 3.9 1.9-3.9 1.9-3.9 128-fold, R.sup.2
= .9813 650/665-X 646 660 26 secs 3.9 3.9 1.9 1.9 256-fold, R.sup.2
= .9288 16-fold, R.sup.2 = .9789 TR-X 588 616 4 secs 7.8 3.9-7.8
3.9 3.9-7.8 128-fold, R.sup.2 = .981 TMR-X 544 570 3 secs 3.9-7.8
3.9 1.9-3.9 3.9 128-fold, R.sup.2 = .9552 FL Br.sub.2 530 545 10
secs 7.8 7.8 3.9-7.8 7.8-15.6 64-fold, R.sup.2 = .9786 R6G 528 547
5 secs 7.8 7.8-15.6 1.9-3.9 15.6 128-fold, R.sup.2 = .9369 64-fold,
R.sup.2 = .9615 FL C.sub.5 504 511 10 secs 7.8 7.8 3.9-7.8 7.8-15.6
32-fold, R.sup.2 = .971 R6G-X 529 547 4 secs 7.8-15.6 3.9-7.8 3.9
3.9 64-fold, R.sup.2 = .9623 FL 502 510 15 secs 7.8-15.6 7.8-15.6
7.8 7.8 16-fold, R.sup.2 = .9872 530/550 534 551 4 secs 7.8-15.6
15.6 3.9-7.8 7.8-15.6 128-fold, R.sup.2 = .9577 493/503 500 509 17
secs 7.8-15.6 15.6-31.25 3.9-7.8 31.25 128-fold, R.sup.2 = .9516
558/568 559 568 3 secs 7.8-15.6 7.8 3.9 3.9-7.8 512-fold, R.sup.2 =
.9645 564/570 563 569 2 secs 3.9-7.8 1.95-3.9 1.95-3.9 7.8
128-fold, R.sup.2 = .9907 576/589 575 588 4 secs 15.6 7.8 3.9-7.8
7.8 128-fold, R.sup.2 = .9922 581/591 581 591 2 secs 7.8-15.6 7.8
3.9 7.8 255-fold, R.sup.2 = .9657 FL-X 504 510 4 secs 3.9-7.8
3.9-7.8 1.9 3.9 256-fold, R.sup.2 = .9722 FITC 494 519 10-15
sec.dagger. 15.6-31.25 125 15.6 31.25 64-fold, R.sup.2 = .9797
32-fold, R.sup.2 = .9936 *Longest exposure without saturation
.dagger.estimated
[0101] As indicated in Table 3, all of the BODIPY dyes tested,
which represent a wide variety of chemical substituents on the core
4-bora-3a,4a-diazaindacene difluoride (dipyrometheneboron
difluoride) core structure provided either superior sensitivity or
greater linearity or both over FITC for detection of all proteins
tested. Furthermore, the BODIPY dyes provided a more uniform
sensitivity for protein detection that was relatively independent
of the protein's structure, a properly not observed with FITC,
which showed considerable differences in sensitivity depending on
the nature of the protein. BODIPY dyes with the additional "X"
(aminohexanoyl) that absorbed maximally between 495 nm and 640 nm
showed particularly high sensitivity, uniformity of staining and
good linear response. Optimal photographic exposure times, which
are another measure of the sensitivity and brightness of the sample
were typically shorter for the BODIPY dyes than for FITC with the
preferred BODIPY dyes having an exposure time of 5 seconds or
less.
29. Simultaneous Dichromatic Detection of Total Protein and a
Specific Target Protein on Microarrays Using BODIPY FL-X, SE and
Wheat Germ Agglutinin.
[0102] PVDF membrane was precut to a 2.5 cm.times.7.5 cm size.
Membranes were made wet by immersing them in 100% methanol followed
by immersion in water for 1 minute. Membranes were then placed on
2.5 cm.times.7.5 cm glass slides held to the surface of the glass
by surface tension. Four specific, purified proteins including C-1
acid glycoprotein, horseradish peroxidase, immunoglobulin G and
soybean trypsin inhibitor were arrayed from a source plate (384
well plate) concentration of 0.0625-4 mg/mL in PBS, onto the PVDF
strips using a manual slide microarrayer. The manual arrayer was
fixed with 4 rows of 8 pins (32 total) with .about.500 micron
diameter spot size, 1.125 micron horizontal pitch and 750 micron
vertical pitch (pitch=center to center spacing of spots). The
proteins were spotted in replicates of 6 resulting in an array of
192 spots including 24 0 ng control spots. After arraying proteins,
the membranes were allowed to dry. To label total proteins the
arrayed membranes were equilibrated in 35 mL of 10 mM sodium borate
buffer, pH 9.5, twice for 10 minutes. After equilibration, the
membranes were stained for 30 minutes with 10 .mu.M BODIPY FL-X,
succinimidyl ester in the same buffer. They were then washed twice
for 2 minutes in 10 mM sodium borate buffer, pH 9.5, three times
for 10 minutes in 100% methanol and finally once for 10 minutes in
dH.sub.2O. Membranes were then allowed to air dry. All steps, not
including equilibration, were performed by placing the membranes in
50 mL centrifuge tubes and placing them on a nutator. Specific
glycoprotein detection was performed by first washing the membranes
for 10 minutes, three times in 50 mM Tris, pH 7.5, 150 mM NaCl
followed by blocking for 1 hour in the same buffer plus 0.25%
MOWIOL 4-88 and 0.2% Tween 20. For detection of glycoproteins, the
membranes were incubated with 1 .mu.g/mL wheat germ
agglutinin-alkaline phosphatase conjugate in a buffer containing 50
mM Tris, pH 7.5, 150 mM NaCl, 0.2% Tween-20, 0.25% MOWIOL 4-88, 0.5
mM MgCl.sub.2, and 1 mM CaCl.sub.2. For detection of the
conjugates, the membranes were incubated in 1.25 g/mL DDAO
phosphate in a buffer containing 1 mM MgCl.sub.2 and 10 mM Tris, pH
9.5. Proteins were viewed using 300 nm UV epi-illumination. Wheat
germ agglutinin binds glycoproteins containing N-acetylglucosamine
and N-acetylneuraminic acid residues. The total protein profile
appeared green fluorescent while the targeted glycoproteins (-1
acid glycoprotein and immunoglobulin G for wheat germ agglutinin)
were stained red fluorescent. Arrays can also be imaged using a
laser system such as the Fuji FLA-3000 imager utilizing the 633 nm
excitation filter and 675 nm emission filter for the DDAO dye and a
473 nm excitation filter and 520 nm emission filter for the BODIPY
FL-X dye. Other BODIPY dyes can be utilized similarly.
30. Simultaneous Dichromatic Detection of Total Protein and a
Specific Target Protein on Microarrays Using BODIPY FL-X, SE and
Concanavalin A.
[0103] Proteins were arrayed, as described in Example 1. To label
total proteins, the arrayed membranes were equilibrated in 35 mL of
10 mM sodium borate buffer, pH 9.5, twice for 10 minutes. After
equilibration, the membranes were stained for 30 minutes with 10
.quadrature.M BODIPY FL-X, succinimidyl ester in the same buffer.
They were washed twice for 2 minutes in 10 mM sodium borate buffer,
pH 9.5, three times for 10 minutes in 100% methanol and finally
once for 10 minutes in dH.sub.2O. Membranes were allowed to air
dry. All steps, not including equilibration, were performed by
placing the membranes in 50 mL centrifuge tubes and placing them on
a nutator. Specific glycoprotein detection was performed by first
washing the membranes for 10 minutes, three times in 50 mM Tris, pH
7.5, 150 mM NaCl followed by blocking for 1 hour in the same buffer
plus 0.25% MOWIOL 4-88 and 0.2% Tween 20. For detection of
glycoproteins, the membranes were incubated with 1 g/mL
concanavalin A-alkaline phosphatase conjugate in a buffer
containing 50 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Tween-20, 0.25%
MOWIOL 4-88, 0.5 mM MgCl.sub.2, and 1 mM CaCl.sub.2. For detection
of the conjugates, the membranes were incubated with 1.25 g/mL DDAO
phosphate in a buffer containing 1 mM MgCl.sub.2 and 10 mM Tris, pH
9.5. Proteins were viewed using 300 nm UV epi-illumination.
Concanavalin A binds to glycoproteins containing - mannosyl and
-glucopyranosyl residues. The total protein profile appeared green
fluorescent while the targeted glycoproteins (horseradish
peroxidase and immunoglobulin G for concanavalin A) were stained
red fluorescent. Arrays can also be imaged using a laser system
such as the Fuji FLA-3000 imager utilizing the 633 nm excitation
filter and 675 nm emission filter for the DDAO dye and a 473 nm
excitation filter and 520 nm emission filter for the BODIPY FL-X
dye. Other BODIPY dyes can be utilized similarly.
31. Simultaneous Dichromatic Detection of Total Protein and a
Specific Target Protein on Microarrays with BODIPY TR-X, SE and
Wheat Germ Agglutinin.
[0104] Proteins were arrayed as described in Example 1. To label
total proteins the arrayed membranes were equilibrated in 35 mL of
10 mM sodium borate buffer, pH 9.5, twice for 10 minutes. After
equilibration, the membranes were stained for 30 minutes in 10 M
BODIPY TR-X, succinimidyl ester in the same buffer. They were then
washed twice for 2 minutes in 10 mM sodium borate buffer, pH 9.5,
three times for 10 minutes in 100% methanol and finally once for 10
minutes in dH.sub.2O. Membranes were allowed to air dry. All steps,
not including equilibration, were performed by placing the
membranes in 50 mL centrifuge tubes and placing them on a nutator.
Specific glycoprotein detection was performed by first washing the
membranes for 10 minutes, three times in 50 mM Tris, pH 7.5, 150 mM
NaCl followed by blocking for 1 hour in the same buffer plus 0.25%
MOWIOL 4-88 and 0.2% Tween 20. For detection of glycoproteins, the
membranes were incubated with 1 g/mL wheat germ
agglutinin-horseradish peroxidase conjugate in a buffer containing
50 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Tween-20, 0.25% MOWIOL 4-88,
0.5 mM MgCl.sub.2, and 1 mM CaCl.sub.2. For detection of the
conjugates, the membranes were incubated with 50 M Amplex Gold in a
buffer containing 10 mM Tris, pH 7.5, 1 mM MgCl.sub.2, 1 mM
ZnCl.sub.2 and 200 M H.sub.2O.sub.2. Proteins were viewed using 300
nm UV epi-illumination. Wheat germ agglutinin binds glycoproteins
containing N-acetylglucosamine and N-acetylneuraminic acid
residues. The total protein profile appeared red fluorescent while
the targeted glycoproteins (-1 acid glycoprotein and immunoglobulin
G for wheat germ agglutinin) were stained gold fluorescent. Arrays
can also be imaged using a laser system such as the Fuji FLA-3000
imager utilizing the 633 nm excitation filter and 675 nm emission
filter for the BODIPY TR-X, SE and a 532 nm excitation filter and
580 nm emission filter for the Amplex Gold. Other BODIPY dyes can
be utilized similarly.
32. Simultaneous Dichromatic Detection of Total Protein and a
Specific Target Protein on Microarrays with BODIPY TR-X, SE and
Concanavalin A.
[0105] Proteins were arrayed, as described in Example 1. To label
total proteins, the arrayed membranes were equilibrated in 35 mL of
10 mM sodium borate buffer, pH 9.5, twice for 10 minutes. After
equilibration, the membranes were stained for 30 minutes with 10 M
BODIPY TR-X, succinimidyl ester in the same buffer. The membranes
were washed twice for 2 minutes in 10 mM sodium borate buffer, pH
9.5, three times for 10 minutes in 100% methanol and finally once
for 10 minutes in dH.sub.2O. Membranes were allowed to air dry. All
steps, not including equilibration, were performed by placing the
membranes in 50 mL centrifuge tubes and placing them on a nutator.
Specific glycoprotein detection was performed by first washing the
membranes for 10 minutes, three times in 50 mM Tris, pH 7.5, 150 mM
NaCl followed by blocking for 1 hour in the same buffer plus 0.25%
MOWIOL 4-88 and 0.2% Tween 20. For detection of glycoproteins, the
membranes were incubated with 1 g/mL concanavalin A-alkaline
phosphatase conjugate in a buffer containing 50 mM Tris, pH 7.5,
150 mM NaCl, 0.2% Tween-20, 0.25% MOWIOL 4-88, 0.5 mM MgCl.sub.2,
and 1 mM CaCl.sub.2. For detection of the conjugates, the membranes
were incubated with 10 g/mL ELF 39 phosphate in a buffer containing
1 mM MgCl.sub.2 and 10 mM Tris, pH 9.5. Proteins were viewed using
300 nm UV epi-illumination. Concanavalin A binds to glycoproteins
containing -mannosyl and -glucopyranosyl residues. The total
protein profile appeared red fluorescent while the targeted
glycoproteins (horseradish peroxidase and immunoglobulin G for
concanavalin A) were stained green fluorescent. Other BODIPY dyes
can be utilized similarly.
* * * * *
References