U.S. patent application number 14/150207 was filed with the patent office on 2014-07-10 for methods and compositions for rapid functional analysis of gene variants.
This patent application is currently assigned to Albert Einstein College of Medicine of Yeshiva University. The applicant listed for this patent is Johnny C. Loke, Harry Ostrer. Invention is credited to Johnny C. Loke, Harry Ostrer.
Application Number | 20140194315 14/150207 |
Document ID | / |
Family ID | 51061403 |
Filed Date | 2014-07-10 |
United States Patent
Application |
20140194315 |
Kind Code |
A1 |
Loke; Johnny C. ; et
al. |
July 10, 2014 |
METHODS AND COMPOSITIONS FOR RAPID FUNCTIONAL ANALYSIS OF GENE
VARIANTS
Abstract
Methods and compositions are disclosed for rapid functional
analysis of gene variants based on analysis of protein-protein and
protein-nucleic acid interactions.
Inventors: |
Loke; Johnny C.; (Nanuet,
NY) ; Ostrer; Harry; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Loke; Johnny C.
Ostrer; Harry |
Nanuet
New York |
NY
NY |
US
US |
|
|
Assignee: |
Albert Einstein College of Medicine
of Yeshiva University
Bronx
NY
|
Family ID: |
51061403 |
Appl. No.: |
14/150207 |
Filed: |
January 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61749960 |
Jan 8, 2013 |
|
|
|
Current U.S.
Class: |
506/9 ;
506/18 |
Current CPC
Class: |
G01N 33/54326 20130101;
G01N 33/6845 20130101 |
Class at
Publication: |
506/9 ;
506/18 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Claims
1. A method of multiplex detecting a first protein-second protein
interaction, in a sample, for up to at least four distinct first
proteins, first proteins A, B, C and D respectively, the method
comprising: contacting the sample with a (i) a first agent attached
to a surface of a magnetic bead that is not labeled with a first
primary optically-active label, and (ii) a second primary agent
attached to a surface of a magnetic bead that is labeled with a
first primary optically-active label, and (iii) a third primary
agent attached to a surface of a non-magnetic bead that is not
labeled with a second primary optically-active label, and (iv) a
fourth primary agent attached to the surface of a non-magnetic bead
that is labeled with a second primary optically-active label,
wherein the first, second, third and fourth primary agents are
different agents each capable of capturing the distinct first
proteins A, B, C and D, respectively; contacting captured first
protein-second protein complex(es) with a plurality of secondary
agents, each of the plurality being specific for a distinct second
protein, and each labeled with a separate secondary
optically-active label wherein the secondary optically-active
labels are not the same as the primary optically-active labels of
the primary agents and are each distinct from the secondary
optically-active label of every other of the optically-active
labeled secondary agents; recovering magnetic beads complexes from
the sample by applying a magnetic field; recovering non-magnetic
bead complexes from the sample based on a non-magnetic physical
property of the non-magnetic beads; passing the recovered magnetic
bead complexes through a flow cytometer or optical plate reader;
passing the recovered non-magnetic bead complexes through a flow
cytometer or optical plate reader; detecting the optical signal(s)
of the recovered magnetic bead complexes; and detecting the optical
signal(s) of the recovered non-magnetic bead complexes; wherein the
presence on a magnetic bead complex of only a secondary
optically-active label indicates the interaction between the first
protein A and a second protein corresponding to the secondary
optically-active labeled secondary agent, and wherein the presence
on a magnetic bead complex of both (i) a first primary
optically-active label and (ii) a secondary optically-active label
indicates the interaction of the first protein B and a second
protein corresponding to the secondary optically-active labeled
secondary agent, and wherein the presence on a non-magnetic bead
complex of only a secondary optically-active label indicates the
interaction of the first protein C and a second protein
corresponding to the secondary optically-active secondary labeled
agent, and wherein the presence on a non-magnetic bead complex of
both (i) a second primary optically-active label and (ii) a
secondary optically-active label indicates the interaction of the
first protein D and a second protein corresponding to the secondary
optically-active labeled secondary agent.
2. A method of multiplex detecting protein-nucleic acid
interactions in a sample for up to at least four distinct proteins,
proteins A, B, C and D respectively, the method comprising: a)
contacting the sample with a (i) a first agent attached to a
surface of a magnetic bead that is not labeled with a first primary
optically-active label, and (ii) a second primary agent attached to
a surface of a magnetic bead that is labeled with a first primary
optically-active label, and (iii) a third primary agent attached to
a surface of a non-magnetic bead that is not labeled with a second
primary optically-active label, and (iv) a fourth primary agent
attached to the surface of a non-magnetic bead that is labeled with
a second primary optically-active label, wherein the first, second,
third and fourth primary agents are different agents each capable
of capturing the distinct proteins A, B, C and D, respectively,
under conditions which permit capturing to the primary agents a
first protein-nucleic acid complex from the sample; b) recovering
magnetic beads complexes from the sample by applying a magnetic
field and recovering non-magnetic bead complexes from the sample
based on a non-magnetic physical property of the non-magnetic
beads; c) contacting one or more of (i) the magnetic bead complexes
not having a first primary optically-active label; (ii) the
magnetic bead complexes having a first primary optically-active
label; (iii) the non-magnetic bead complexes not having a first
primary optically-active label; (iv) the non-magnetic bead
complexes having a first primary optically-active label, with a
Proteinase K so as to digest the proteins thereon and release any
nucleic acids bound thereto; d) sequencing nucleic acid(s) released
in step c)(i) so as to thereby identify the nucleic acids that have
interacted with distinct protein A; in step c)(ii) so as to thereby
identify the nucleic acids that have interacted with distinct
protein B; in step c)(iii) so as to thereby identify the nucleic
acids that have interacted with distinct protein C; and/or in step
c)(iv) so as to thereby identify the nucleic acids that have
interacted with distinct protein D.
3. The method of claim 2, further comprising probing the
protein-nucleic acid complex(es) with one or more optically active
secondary agents each specific for one of distinct proteins A, B, C
and D, so as to identify bead complexes comprising a bead, a
distinct protein and a primary agent, and recovering such bead
complexes.
4. The method of claim 3, further comprising after step c) and
before step d) passing the recovered magnetic bead complexes
through a flow cytometer or optical plate reader and passing the
recovered non-magnetic bead complexes through a flow cytometer or
optical plate reader; and detecting the optical signal(s) of the
recovered magnetic bead complexes and detecting the optical
signal(s) of the recovered non-magnetic bead complexes and,
optionally, quantifying the optical signal(s) detected so as to
thereby quantify the amount of protein-nucleic acid interaction on
the bead.
5. The method of claim 2, further comprising amplifying the nucleic
acids released after contacting with a Proteinase K, but prior to
sequencing.
6. The method of claim 1, wherein the presence on a bead complex of
a first primary optically-active label and/or a secondary
optically-active label is determined by quantifying the optical
signal thereof.
7. The method of claim 5, wherein the optical signal is collected
with one or more photomultipliers.
8. The method of claim 1, further comprising quantifying the
optical signal(s) detected so as to thereby quantify the amount of
first protein-second protein interaction on the bead and,
optionally, comparing the quantified amount against a control
amount or control curve.
9. The method of claim 1, wherein each primary agent comprises an
antibody or comprises an antigen-binding fragment of an
antibody.
10. The method of claim 1, wherein each secondary agent comprises
an antibody or comprises an antigen-binding fragment of an
antibody.
11. The method of claim 1, wherein the sample is a cell or tissue
lysate.
12-15. (canceled)
16. The method of claim 1, wherein FSC and/or SSC are adjusted with
a control un-complexed bead population prior to initiating the
method so as to permit complexed beads to be detected.
17-18. (canceled)
19. The method of claim 9, wherein the antibodies are monoclonal
antibodies.
20. The method of claim 9, wherein the antibody fragments are
F(ab').sub.2 fragments, Fab' fragments or ScFvs.
21. The method of claim 1, wherein the magnetic beads are
epoxy-coated magnetic beads.
22. The method of claim 1, wherein the non-magnetic beads are
carboxyl modified beads.
23-27. (canceled)
28. The method of claim 1, wherein forward scatter amplitude gain
and side scatter voltage on a flow cytometer are set to register
populations of bead events to on scale, followed by applying an
inclusion gate where selected linear populations of beads form
collective clusters containing interrogation targets can be
analyzed in their entirety by flow cytometry.
29. (canceled)
30. A kit for detecting changes in protein expression in cells and
for analysis of gene variants, the kit comprising: magnetic beads
for immunoprecipitation, non-magnetic beads for
immunoprecipitation, a lysis formulation, one or more Proteinase K
inhibitors, one or more phosphatase inhibitors, a coupling buffer,
nucleic acids recovery elution buffer, one or more functional
variant assay (FVA) buffers, a Western loading buffer, one or more
optically active labels, and instructions for use of the kit.
31-36. (canceled)
37. A kit for obtaining nuclear, cytoplasmic or whole-cell extract
from cells or from tissue, the kit comprising: cell lysis buffer 1M
Dithiothreitol (DTT) one or more phosphatase inhibitors
10.times.PBS one or more phosphatase inhibitors 10.times. hypotonic
buffer detergent written instructions for use of the kit.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 61/749,960, filed Jan. 8, 2013, the contents of
which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to methods and compositions for rapid
functional analysis of gene variants based on protein-protein and
protein-nucleic acid interactions.
BACKGROUND OF THE INVENTION
[0003] Throughout this application various publications are
referred to in parentheses. Full citations for these references may
be found at the end of the specification before the claims. The
disclosures of these publications are hereby incorporated by
reference in their entireties into the subject application to more
fully describe the art to which the subject application
pertains.
[0004] The availability of accurate, relatively low-cost sequencing
methods to analyze the exome or whole genome for novel, rare
variants that affect phenotypes has become a game changer in
clinical genetics (1). Deciphering the newly identified variants is
not a simple task. For any individual genome, up to 3.5 million
single nucleotide variants and 600,000 indels may be identified
(2). Similarly for any given exome, up to 17,000 variants may be
identified (3). To reduce the complexity of analysis, variants are
filtered using bioinformatics for rarity by comparison with
catalogs, such as dbSNP and 1000 genomes (3-4). Yet, observing a
hit in these catalogs does not negate a possible phenotypic effect
biologically. Current gold standard computational methods, such as
NNSPLICE that predicts splicing alteration (5), and SIFT, SNAP and
PolyPhen that predict possible deleterious effects based on
conservation of encoded amino acids may also fall short for both
sensitivity and specificity (6-8). Linkage, homozygosity mapping
and other purely genetic methods may lack statistical power from
limited number of affected individuals within a pedigree or
community available for study. Furthermore, demonstration of
linkage even at a very high LOD score does not preclude the
presence of a second variant in linkage disequilibrium that is in
fact causal. The observation that 85% of previously identified
causal variants for monogenic disorders were identified in exons or
at splice-junction boundaries in introns strongly suggests that the
vast majority affect the quantity and/or function of the encoded
gene RNA and/or protein products (9). In addition, most active
proteins are members of multimeric complexes (10). Thus, mutations
in a candidate gene may change the quantity of the protein that it
encodes, may alter the post-translational modification of that
protein or may affect its interaction and localization with its
crucial protein binding partners altogether. All three of these
alterations can be assessed by immunoassays, which have been a
mainstay for quantifying unmodified and modified proteins for over
30 years (11). These methods include both immunohistochemical
studies of cells and Western blots of cell homogenates. Dual
immunoassays, such as those provided by co-immunoprecipitation
(co-IP) followed by Western blots have become important for
quantifying protein-protein interactions as functional studies
(12).
[0005] The present invention addresses the need for rapid
functional analysis of gene variants for phenotype effects based on
protein-protein and protein-nucleic acid interactions and
localization.
SUMMARY OF THE INVENTION
[0006] A method is provided for multiplex detecting a first
protein-second protein interaction, in a sample, for up to four
distinct first proteins, first proteins A, B, C and D respectively,
the method comprising:
contacting the sample with a (i) a first agent attached to a
surface of a magnetic bead that is not labeled with a first primary
optically-active label, and (ii) a second primary agent attached to
a surface of a magnetic bead that is labeled with a first primary
optically-active label, and (iii) a third primary agent attached to
a surface of a non-magnetic bead that is not labeled with a second
primary optically-active label, and (iv) a fourth primary agent
attached to the surface of a non-magnetic bead that is labeled with
a second primary optically-active label, wherein the first, second,
third and fourth primary agents are different agents each capable
of capturing the distinct first proteins A, B, C and D,
respectively;
[0007] contacting captured first protein-second protein complex(es)
with a plurality of secondary agents each specific for a distinct
second protein, and each labeled with a separate secondary
optically-active label wherein the secondary optically-active
labels are not the same as the primary optically-active labels of
the primary agents and are each distinct from the secondary
optically-active label of every other of the optically-active
labeled secondary agents;
[0008] recovering magnetic beads complexes from the sample by
applying a magnetic field;
[0009] recovering non-magnetic bead complexes from the sample based
on a non-magnetic physical property of the non-magnetic beads;
[0010] passing the recovered magnetic bead complexes through a flow
cytometer or optical plate reader;
[0011] passing the recovered non-magnetic bead complexes through a
flow cytometer or optical plate reader;
[0012] detecting the optical signal(s) of the recovered magnetic
bead complexes; and
[0013] detecting the optical signal(s) of the recovered
non-magnetic bead complexes;
[0014] wherein the presence on a magnetic bead complex of only a
secondary optically-active label indicates the interaction between
the first protein A and a second protein corresponding to the
secondary optically-active labeled secondary agent,
[0015] and wherein the presence on a magnetic bead complex of both
(i) a first primary optically-active label and (ii) a secondary
optically-active label indicates the interaction of the first
protein B and a second protein corresponding to the secondary
optically-active labeled secondary agent,
[0016] and wherein the presence on a non-magnetic bead complex of
only a secondary optically-active label indicates the interaction
of the first protein C and a second protein corresponding to the
secondary optically-active secondary labeled agent,
[0017] and wherein the presence on a non-magnetic bead complex of
both (i) a second primary optically-active label and (ii) a
secondary optically-active label indicates the interaction of the
first protein D and a second protein corresponding to the secondary
optically-active labeled secondary agent.
[0018] Also provided is a method of multiplex detecting a first
protein-second protein interaction, in a sample, for up to at least
four distinct first proteins, first proteins A, B, C and D
respectively, the method comprising:
[0019] contacting the sample with a (i) a first agent attached to a
surface of a magnetic bead that is not labeled with a first primary
optically-active label, and (ii) a second primary agent attached to
a surface of a magnetic bead that is labeled with a first primary
optically-active label, and (iii) a third primary agent attached to
a surface of a non-magnetic bead that is not labeled with a second
primary optically-active label, and (iv) a fourth primary agent
attached to the surface of a non-magnetic bead that is labeled with
a second primary optically-active label,
wherein the first, second, third and fourth primary agents are
different agents each capable of capturing the distinct first
proteins A, B, C and D, respectively;
[0020] contacting captured first protein-second protein complex(es)
with a plurality of secondary agents, each of the plurality being
specific for a distinct second protein, and each labeled with a
separate secondary optically-active label wherein the secondary
optically-active labels are not the same as the primary
optically-active labels of the primary agents and are each distinct
from the secondary optically-active label of every other of the
optically-active labeled secondary agents;
[0021] recovering magnetic beads complexes from the sample by
applying a magnetic field;
[0022] recovering non-magnetic bead complexes from the sample based
on a non-magnetic physical property of the non-magnetic beads;
[0023] passing the recovered magnetic bead complexes through a flow
cytometer or optical plate reader;
[0024] passing the recovered non-magnetic bead complexes through a
flow cytometer or optical plate reader;
[0025] detecting the optical signal(s) of the recovered magnetic
bead complexes; and
[0026] detecting the optical signal(s) of the recovered
non-magnetic bead complexes;
[0027] wherein the presence on a magnetic bead complex of only a
secondary optically-active label indicates the interaction between
the first protein A and a second protein corresponding to the
secondary optically-active labeled secondary agent,
[0028] and wherein the presence on a magnetic bead complex of both
(i) a first primary optically-active label and (ii) a secondary
optically-active label indicates the interaction of the first
protein B and a second protein corresponding to the secondary
optically-active labeled secondary agent,
[0029] and wherein the presence on a non-magnetic bead complex of
only a secondary optically-active label indicates the interaction
of the first protein C and a second protein corresponding to the
secondary optically-active secondary labeled agent,
[0030] and wherein the presence on a non-magnetic bead complex of
both (i) a second primary optically-active label and (ii) a
secondary optically-active label indicates the interaction of the
first protein D and a second protein corresponding to the secondary
optically-active labeled secondary agent.
[0031] Also provided is a method of multiplex detecting a
protein-nucleic acid interaction, in a sample, for up to four
distinct proteins, proteins A, B, C and D respectively, the method
comprising:
[0032] contacting the sample with a (i) a first agent attached to a
surface of a magnetic bead that is not labeled with a first primary
optically-active label, and (ii) a second primary agent attached to
a surface of a magnetic bead that is labeled with a first primary
optically-active label, and (iii) a third primary agent attached to
a surface of a non-magnetic bead that is not labeled with a second
primary optically-active label, and (iv) a fourth primary agent
attached to the surface of a non-magnetic bead that is labeled with
a second primary optically-active label,
wherein the first, second, third and fourth primary agents are
different agents each capable of capturing the distinct first
proteins A, B, C and D, respectively, under conditions which permit
capturing to the primary agents a first protein-nucleic acid
complex from the sample;
[0033] contacting captured first protein-nucleic acid complex(es)
with a plurality of secondary agents each specific for a distinct
nucleic acid, and each labeled with a separate secondary
optically-active label wherein the secondary optically-active
labels are not the same as the primary optically-active labels of
the primary agents and are each distinct from the secondary
optically-active label of every other optically-active labeled
secondary agent;
[0034] recovering magnetic beads complexes from the sample by
applying a magnetic field;
[0035] recovering non-magnetic bead complexes from the sample based
on a non-magnetic physical property of the non-magnetic beads;
[0036] passing the recovered magnetic bead complexes through a flow
cytometer or optical plate reader;
[0037] passing the recovered non-magnetic bead complexes through a
flow cytometer or optical plate reader;
[0038] quantifying the optical signal(s) of the recovered magnetic
bead complexes; and
[0039] quantifying the optical signal(s) of the recovered
non-magnetic bead complexes;
[0040] wherein the presence on a magnetic bead complex of only a
secondary optically-active label indicates the interaction between
the first protein A and a nucleic acid corresponding to the
secondary optically-active labeled secondary agent,
[0041] and wherein the presence on a magnetic bead complex of both
(i) a first primary optically-active label and (ii) a secondary
optically-active label indicates the interaction of the first
protein B and a nucleic acid corresponding to the secondary
optically-active labeled secondary agent,
[0042] and wherein the presence on a non-magnetic bead complex of
only a secondary optically-active label indicates the interaction
of the first protein C and a nucleic acid corresponding to the
secondary optically-active secondary labeled agent,
[0043] and wherein the presence on a non-magnetic bead complex of
both (i) a second primary optically-active label and (ii) a
secondary optically-active label indicates the interaction of the
first protein D and a nucleic acid corresponding to the secondary
optically-active labeled secondary agent.
[0044] The invention provides methods of analyzing a gene variant
based on endogeneous or transient protein-protein interaction, the
method comprising: attaching a first primary agent to the surface
of magnetic beads that are not labeled with an optically active
label, such as for example, but not limited to, a fluorescent
label, and attaching a second primary agent to the surface of
magnetic beads that are labeled with an optically active label;
attaching a third primary agent to the surface of non-magnetic
beads that are not labeled with an optically active label and
attaching a fourth primary agent to the surface of non-magnetic
beads that are labeled with an optically active label, wherein the
first, second, third and fourth primary agents are different agents
and wherein the first, second, third and fourth primary agents are
each capable of capturing a distinct protein complex from a cell or
tissue lysate; capturing to the primary agents a protein complex
from a cell or tissue lysate, where the protein complex comprises a
protein of interest that is a product of a gene or a gene variant
and where the protein of interest is part of a complex with another
protein; probing the protein-protein complex with one or more
optically active labeled secondary agents specific for a member of
the complex; wherein the same one or more optically active labels
can be used to label secondary agents on any of i) the magnetic
beads that are not labeled with an optically active label, ii) the
magnetic beads that are labeled with an optically active label,
iii) the non-magnetic beads that are not labeled with an optically
active label, and iv) the non-magnetic beads that are labeled with
an optically active label; separating magnetic beads from the
lysate based on magnetic properties of the magnetic beads;
separating non-magnetic beads from the lysate based on a physical
property of the non-magnetic beads; and measuring optical activity
of optically active agents, wherein the absence or presence of the
optically active label on the magnetic beads is used as an
identifier to distinguish optically active protein complexes
captured by the first and second primary agents, respectively, and
wherein the absence or presence of the optically active label on
the non-magnetic beads is used as an identifier to distinguish
optically active protein complexes captured by the third and fourth
primary agents, respectively.
[0045] The invention also provides methods of analyzing a gene
variant based on endogenous or transient protein-nucleic acid
interaction, the method comprising: attaching a first primary agent
to the surface of magnetic beads that are not labeled with an
optically active label and attaching a second primary agent to the
surface of magnetic beads that are labeled with an optically active
label; attaching a third primary agent to the surface of
non-magnetic beads that are not labeled with an optically active
label and attaching a fourth primary agent to the surface of
non-magnetic beads that are labeled with an optically active label,
wherein the first, second, third and fourth primary agents are
different agents and wherein the first, second, third and fourth
primary agents are each capable of capturing a distinct
protein-nucleic acid complex from a cell or tissue lysate;
capturing to the primary agents a protein-nucleic acid complex from
a cell or tissue lysate, where the protein-nucleic acid complex
comprises a gene or a gene variant nucleic acid sequence;
separating magnetic beads from the lysate based on magnetic
properties of the magnetic beads; separating non-magnetic beads
from the lysate based on a physical property of the non-magnetic
beads; optionally digesting nucleic acids with nucleases prior to
digesting proteins on protein-nucleic acid complexes to release
nucleic acids; and amplifying the released nucleic acids; wherein
the absence or presence of the optically active label on the
magnetic beads is used to distinguish optically active
protein-nucleic complexes captured by the first and second primary
agents, respectively, and wherein the absence or presence of the
optically active label on the non-magnetic beads is used to
distinguish optically active protein-nucleic acid complexes
captured by the third and fourth primary agents, respectively.
[0046] The invention effectively incorporate methods for optimal
detection and selection of various targets (more than two) with
limited starting biomaterial. The invention includes a gating
principle that substantially increases sensitivity of the detection
of flow cytometry-based immunoassay with no amplification steps of
any sort.
[0047] The invention provides kits for identification of the
protein-protein interactions, protein-nucleic acid interactions,
cell-based protein expression, protein modifications, localization
and a standard concurrent immunoprecipitation (IP)-Western. The
kits allow a simplified flow-based immunoassay that is truly
high-throughput and unified sample processing techniques. The kits
contain optimized comprehensive chemistries and significant
improvement over traditional methods of IP-Western blots.
[0048] The kit provides versatility to perform multiple assays,
assays such as Digital Cell Western (DCW), rapid assessment of
protein-protein interaction and localization by modified flow
cytometry-based IP, simplified protein-nucleic acid interactions
assessment and captured nucleic acid purification for massive
parallel sequencing (MPS), genotyping and polymerase chain
reactions (PCR) applications.
[0049] DCW, a form of digital Western in this kit that is designed
and optimized to probe individual cells for various target protein
expressions including post-translational modifications that can be
effortlessly detected simultaneously allowing thousands of data
points from each fixed cell samples to be aggregated as digital
calculation for statistical power.
[0050] The invention utilizes a combination of bar-coded bead
system, for example but not limited to surface enhancedDynabeads
and Carboxyl modified beads (CML) to detect multiple variant
protein interactions from a single lysate. With the paramagnetic
properties of Dynabeads, first separation phase allows clearance of
targets bound to the Dynabeads by magnetic separation, remaining
supernatant containing CML beads will capture another set of
targets that will be separated by centrifugal force. This approach
using bar-coded bead systems allows doubling of the number of
detected targets with this methodology in various high-throughput
formats.
[0051] The invention allows assessment of protein-nucleic acid
interactions and purification of bound nucleic acids sequences,
which is suitable for use in Chromatin Immunoprecipitation (CHIP),
RNA IP PCR, genotyping and sequencing (MPS). This is first of its
kind kit that allows assessment of variant protein interactions and
simultaneous purification of any binding nucleic acid sequences to
the variant proteins. Applications such as CHIP, CHIP-sequencing,
and RNA IP are transformed into a streamlined assay suitable for
large-scaled investigation of various targets in research and
diagnostic applications.
[0052] Also provided is a method of multiplex detecting a
protein-nucleic acid interaction, in a sample, for up to at least
four distinct proteins, proteins A, B, C and D respectively, the
method comprising:
[0053] a) contacting the sample with a (i) a first agent attached
to a surface of a magnetic bead that is not labeled with a first
primary optically-active label, and (ii) a second primary agent
attached to a surface of a magnetic bead that is labeled with a
first primary optically-active label, and (iii) a third primary
agent attached to a surface of a non-magnetic bead that is not
labeled with a second primary optically-active label, and (iv) a
fourth primary agent attached to the surface of a non-magnetic bead
that is labeled with a second primary optically-active label,
wherein the first, second, third and fourth primary agents are
different agents each capable of capturing the distinct first
proteins A, B, C and D, respectively, under conditions which permit
capturing to the primary agents a first protein-nucleic acid
complex from the sample;
[0054] b) optionally, contacting one or more of the four distinct
proteins of the sample with one or more nucleic acids either prior
to a) or subsequent to a);
[0055] c) recovering magnetic beads complexes from the sample by
applying a magnetic field and recovering non-magnetic bead
complexes from the sample based on a non-magnetic physical property
of the non-magnetic beads;
[0056] d) contacting one or more of (i) the magnetic beads
complexes not having a first primary optically-active label; (ii)
the magnetic beads complexes having a first primary
optically-active label; (iii) the non-magnetic beads complexes not
having a first primary optically-active label; (iv) the
non-magnetic beads complexes having a first primary
optically-active label, with optional nuclease thena Proteinase K
so as to digest the proteins thereon and release any nucleic acids
bound thereto;
[0057] e) sequencing nucleic acid(s) released in step d)(i) so as
to thereby identify the nucleic acids that have interacted with the
first distinct protein; in step d)(ii) so as to thereby identify
the nucleic acids that have interacted with the second distinct
protein; in step d)(iii) so as to thereby identify the nucleic
acids that have interacted with the third distinct protein; and/or
in step d)(iv) so as to thereby identify the nucleic acids that
have interacted with the fourth distinct protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIG. 1. Application areas of Functional Variant Assay (FVA)
kit. The kit provides versatility using flow cytometry to perform
multiple assays, such as Digital Cell Western (DCW) concurrent with
traditional IP western, rapid assessment of protein-protein
interactions of various target using limited biomaterial,
protein-nucleic acid interactions assessment, bound nucleic acid
purification and enrichment for MPS, genotyping and polymerase
chain reactions (PCR) applications. DCW is a form of digital
western in this kit that is designed and optimized to probe
individual cells for various target protein expressions including
post-translational modifications can be effortlessly detected,
while simultaneously measuring thousands of data points from each
of the fixed cell samples; data points then can be aggregated as
digital calculations for statistical power.
[0059] FIG. 2. Examples of application of the invention. The
invention allows assessment by modified flow immunoprecipitation of
protein-protein interactions, protein-nucleic acid interactions and
purification with enrichment of bound/captured nucleic acids
sequences suitable for use in PCR, genotyping and sequencing (MPS).
The kit allows concurrent assessment of variant protein
interactions, digital cell western and simultaneous purification of
any binding nucleic acid sequences to the variant proteins for MPS.
Applications such as Chromatin Immunoprecipitation (CHIP),
CHIP-sequencing, and RNA IP are transformed into a streamlined
assay suitable for large-scaled investigation of various targets in
research and diagnostic applications.
[0060] FIG. 3. Example of scheme of the present invention. The
invention utilizes a combination of beads, for example but not
limited to epoxy Dynabeads and Carboxyl modified beads (CML) to
detect multiple variant proteins. With the paramagnetic properties
of Dynabeads, a first separation phase allows clearance of targets
bound to the Dynabeads by magnetic separation, remaining
supernatant containing CML beads will capture another set of
targets that will be separated by centrifugal force. This approach
using two-bead system will double the number of detected targets
using existing methodology optimized for high-throughput analysis.
Bar coding complexes to identify different protein-protein
complexes or different protein-nucleic acid complexes on beads with
different optically active labels ("vertical bar coding"). Another
form of bar-coding means to identify different analytes on the
magnetic beads and/or non-magnetic beads using optically active
labels, for example but not limited to fluorescent labels
("horizontal bar coding"). For example but not limited to, for 7
different fluorescent labels, one label can be used to identify a
portion and/or subset of magnetic or non-magnetic beads and the
remaining 6 labels can be used to label different protein targets
or different protein-nucleic acid complexes attached to the beads.
This method is expandable depending on the capabilities and the
number of available lasers on the flow cytometer or plate
readers.
[0061] FIG. 4A-4C. Functional Variant Assay (FVA) performed on
B-lymphoblastoid cells from wild-type and p.Leu189Arg using the
MAP3K1 bait antibody and the Alexa 647 and Alexa 488-labeled MAP3K4
target antibodies. A. The flow cytometry gated results shows
increased binding of MAP3K4 to mutant MAP3K1 as shown previously by
standard methods. B. Results compiled from three independent
experiments for each pathogenic mutation, Leu189Arg, p.Leu189Pro
and c.634-8A show increased MAP3K4 binding to mutant MAP3K1
(p<0.05). C. Conventional IP Western blots of primary
B-lymphoblastoid cells detected an approximate 2-fold increase of
binding of MAP3K4 to MAP3K1 from all three mutant cell lines
compared to wild-type. Loading control is actin and input control
MAP3K1 on the lowest panel.
[0062] FIG. 5A-5C. Reverse FVA performed on B-lymphoblastoid cells
from wild-type and p.Leu189Arg using the RHOA bait antibody and the
Alexa 488-labeled MAP3K1 target antibody. A. The flow cytometry
gated results shows increased binding of mutant MAP3K1 to RHOA
complexes. B. Results compiled from three independent experiments
for each pathogenic mutation, Leu189Arg, p.Leu189Pro and c.634-8A,
show increased mutant MAP3K1 binding to RHOA (p<0.05). C.
Conventional IP Western blots of primary B-lymphoblastoid cells
detect an approximate 2.5-fold increase of binding of mutant MAP3K1
to RHOA from all three mutant cell lines compared to wild-type.
[0063] FIG. 6A-6C. A. Individual sample intensities of MAP3K1 input
prior to pull-down for MAP3K4 for FIG. 4C were quantified from
conventional Western blots using the Licor Software 3.0. B. After
controlling for MAP3K1 loading the intensities were further
normalized to actin, showing an average 2-fold increase of MAP3K4
binding in all mutant samples. C. Reverse IP using RHOA as bait
shows increased binding of MAP3K1 to all mutant samples, about
2.5-fold increase compared to WT samples. These results were
normalized to histone as a loading control.
[0064] FIG. 7A-7B. A. After treatment with Etoposide and UV
radiation or B. the X-Ray mimetic drug, Bleomycin, the localization
of BRCA 1 to nuclear foci was markedly lower among mutant samples
compared to normal samples as measured with the present assay.
[0065] FIG. 8. Traditional phospho-Western vs. Digital Cell
phospho-Western. Preparation and run: 2 days vs. less than 2 hours.
Traditional IP-western vs. Flow Variant Analysis: Preparation and
run time: 5 days vs. 5 hours.
DETAILED DESCRIPTION OF THE INVENTION
[0066] A method is provided of multiplex detecting a first
protein-second protein interaction, in a sample, for up to at least
four distinct first proteins, first proteins A, B, C and D
respectively, the method comprising:
[0067] contacting the sample with a (i) a first agent attached to a
surface of a magnetic bead that is not labeled with a first primary
optically-active label, and (ii) a second primary agent attached to
a surface of a magnetic bead that is labeled with a first primary
optically-active label, and (iii) a third primary agent attached to
a surface of a non-magnetic bead that is not labeled with a second
primary optically-active label, and (iv) a fourth primary agent
attached to the surface of a non-magnetic bead that is labeled with
a second primary optically-active label,
wherein the first, second, third and fourth primary agents are
different agents each capable of capturing the distinct first
proteins A, B, C and D, respectively;
[0068] contacting captured first protein-second protein complex(es)
with a plurality of secondary agents each specific for a distinct
second protein, and each labeled with a separate secondary
optically-active label wherein the secondary optically-active
labels are not the same as the primary optically-active labels of
the primary agents and are each distinct from the secondary
optically-active label of every other of the optically-active
labeled secondary agents;
[0069] recovering magnetic beads complexes from the sample by
applying a magnetic field;
[0070] recovering non-magnetic bead complexes from the sample based
on a non-magnetic physical property of the non-magnetic beads;
[0071] passing the recovered magnetic bead complexes through a flow
cytometer or optical plate reader and quantifying the optical
signal emitted therefrom;
[0072] passing the recovered non-magnetic bead complexes through a
flow cytometer or optical plate reader and quantifying the optical
signal emitted therefrom;
[0073] detecting the optical signal(s) of the recovered magnetic
bead complexes; and
[0074] detecting the optical signal(s) of the recovered
non-magnetic bead complexes;
[0075] wherein the presence on a magnetic bead complex of only a
secondary optically-active label indicates the interaction between
the first protein A and a second protein corresponding to the
secondary optically-active labeled secondary agent,
[0076] and wherein the presence on a magnetic bead complex of both
(i) a first primary optically-active label and (ii) a secondary
optically-active label indicates the interaction of the first
protein B and a second protein corresponding to the secondary
optically-active labeled secondary agent,
[0077] and wherein the presence on a non-magnetic bead complex of
only a secondary optically-active label indicates the interaction
of the first protein C and a second protein corresponding to the
secondary optically-active secondary labeled agent,
and wherein the presence on a non-magnetic bead complex of both (i)
a second primary optically-active label and (ii) a secondary
optically-active label indicates the interaction of the first
protein D and a second protein corresponding to the secondary
optically-active labeled secondary agent.
[0078] A method of multiplex detecting a first protein-second
protein interaction, in a sample, for up to at least four distinct
first proteins, first proteins A, B, C and D respectively, the
method comprising:
[0079] contacting the sample with a (i) a first agent attached to a
surface of a magnetic bead that is not labeled with a first primary
optically-active label, and (ii) a second primary agent attached to
a surface of a magnetic bead that is labeled with a first primary
optically-active label, and (iii) a third primary agent attached to
a surface of a non-magnetic bead that is not labeled with a second
primary optically-active label, and (iv) a fourth primary agent
attached to the surface of a non-magnetic bead that is labeled with
a second primary optically-active label,
wherein the first, second, third and fourth primary agents are
different agents each capable of capturing the distinct first
proteins A, B, C and D, respectively;
[0080] contacting captured first protein-second protein complex(es)
with a plurality of secondary agents, each of the plurality being
specific for a distinct second protein, and each labeled with a
separate secondary optically-active label wherein the secondary
optically-active labels are not the same as the primary
optically-active labels of the primary agents and are each distinct
from the secondary optically-active label of every other of the
optically-active labeled secondary agents;
[0081] recovering magnetic beads complexes from the sample by
applying a magnetic field;
[0082] recovering non-magnetic bead complexes from the sample based
on a non-magnetic physical property of the non-magnetic beads;
[0083] passing the recovered magnetic bead complexes through a flow
cytometer or optical plate reader;
[0084] passing the recovered non-magnetic bead complexes through a
flow cytometer or optical plate reader;
[0085] detecting the optical signal(s) of the recovered magnetic
bead complexes; and
[0086] detecting the optical signal(s) of the recovered
non-magnetic bead complexes;
[0087] wherein the presence on a magnetic bead complex of only a
secondary optically-active label indicates the interaction between
the first protein A and a second protein corresponding to the
secondary optically-active labeled secondary agent,
[0088] and wherein the presence on a magnetic bead complex of both
(i) a first primary optically-active label and (ii) a secondary
optically-active label indicates the interaction of the first
protein B and a second protein corresponding to the secondary
optically-active labeled secondary agent,
[0089] and wherein the presence on a non-magnetic bead complex of
only a secondary optically-active label indicates the interaction
of the first protein C and a second protein corresponding to the
secondary optically-active secondary labeled agent,
[0090] and wherein the presence on a non-magnetic bead complex of
both (i) a second primary optically-active label and (ii) a
secondary optically-active label indicates the interaction of the
first protein D and a second protein corresponding to the secondary
optically-active labeled secondary agent.
[0091] Such methods may be termed "PrCo-IP." In an embodiment, the
plurality of secondary agents is up to twelve secondary agents. In
an embodiment, the plurality of secondary agents is up to
twenty-four secondary agents. In an embodiment, the plurality of
secondary agents is up to thirty-six secondary agents. In an
embodiment, the plurality of secondary agents is, not limited to,
up to thirty-eight secondary agents. The method may be performed
with as many types of secondary agents as are discretely
distinguishable.
[0092] The method can further comprise multiplex detecting more
than four distinct proteins. For detecting n distinct proteins, the
magnetic and non-magnetic bead populations must comprise between
them a primary agent for each of the n proteins and at least n-2
primary optically-active labels, one for each of n-2 of the
proteins. The remaining 2 proteins can be detected by the magnetic
and non-magnetic beads which having the primary agent for each of
those proteins, but which are unlabeled with the primary
optically-active agents.
[0093] A method of multiplex detecting protein-nucleic acid
interactions in a sample for up to four distinct proteins, proteins
A, B, C and D respectively, the method comprising:
[0094] a) contacting the sample with a (i) a first agent attached
to a surface of a magnetic bead that is not labeled with a first
primary optically-active label, and (ii) a second primary agent
attached to a surface of a magnetic bead that is labeled with a
first primary optically-active label, and (iii) a third primary
agent attached to a surface of a non-magnetic bead that is not
labeled with a second primary optically-active label, and (iv) a
fourth primary agent attached to the surface of a non-magnetic bead
that is labeled with a second primary optically-active label,
wherein the first, second, third and fourth primary agents are
different agents each capable of capturing the distinct proteins A,
B, C and D, respectively, under conditions which permit capturing
to the primary agents a first protein-nucleic acid complex from the
sample;
[0095] b) recovering magnetic beads complexes from the sample by
applying a magnetic field and recovering non-magnetic bead
complexes from the sample based on a non-magnetic physical property
of the non-magnetic beads;
[0096] c) contacting one or more of (i) the magnetic bead complexes
not having a first primary optically-active label; (ii) the
magnetic bead complexes having a first primary optically-active
label; (iii) the non-magnetic bead complexes not having a first
primary optically-active label; (iv) the non-magnetic bead
complexes having a first primary optically-active label, with a
Proteinase K so as to digest the proteins thereon and release any
nucleic acids bound thereto;
[0097] d) sequencing nucleic acid(s) released in step c)(i) so as
to thereby identify the nucleic acids that have interacted with
distinct protein A; in step c)(ii) so as to thereby identify the
nucleic acids that have interacted with distinct protein B; in step
c)(iii) so as to thereby identify the nucleic acids that have
interacted with distinct protein C; and/or in step c)(iv) so as to
thereby identify the nucleic acids that have interacted with
distinct protein D.
[0098] Such a method may be termed "NACo-IP." In an embodiment, the
method further comprises contacting one or more of the four
distinct proteins of the sample with one or more nucleic acids
either prior to a) or subsequent to a). It is apparent that in some
circumstances the sample will contain proteins-nucleic acid
interactions prior to application of the method in which case such
a step is not required. In other cases, where the protein(s) of the
sample are free of nucleic interaction prior to application of the
method, nucleic acids can be contacted with the proteins to
determine if the nucleic acids interact with the proteins. In an
embodiment, the plurality of secondary agents is up to twelve
secondary agents. In an embodiment, the plurality of secondary
agents is up to twenty-four secondary agents. In an embodiment, the
plurality of secondary agents is up to thirty-six secondary agents.
In an embodiment, the plurality of secondary agents is up to
thirty-eight secondary agents. The method may be performed with as
many types of secondary agents as are discretely
distinguishable.
[0099] The method can further comprise multiplex detecting more
than four distinct protein-nucleic acid interactions. For detecting
n distinct protein-nucleic acid interactions, the magnetic and
non-magnetic bead populations must comprise between them a primary
agent for each of the n proteins.
[0100] In an embodiment, the method further comprises digesting the
proteins of the protein-nucleic acids with a Proteinase K. In an
embodiment, the method further comprises amplifying the nucleic
acids released after Proteinase K digestion. In an embodiment, the
method is used to identify genetic heterogeneity among a population
of cells. In an embodiment, the sample contacted with the primary
agents is subsequently contacted with a nuclease to digest nucleic
acids that are not interacting/bound to a protein of the sample. In
an embodiment, the method further comprises probing the
protein-nucleic acid complex(es) with one or more optically active
secondary agents each specific for one of distinct first proteins
A, B, C and D, so as to identify bead complexes comprising a bead,
a distinct protein and a primary agent, and recovering such bead
complexes. In an embodiment, the method further comprises
comprising after step c) and before step d) passing the recovered
magnetic bead complexes through a flow cytometer or optical plate
reader and passing the recovered non-magnetic bead complexes
through a flow cytometer or optical plate reader; and detecting the
optical signal(s) of the recovered magnetic bead complexes and
detecting the optical signal(s) of the recovered non-magnetic bead
complexes and, optionally, quantifying the optical signal(s)
detected so as to thereby quantify the amount of protein-nucleic
acid interaction on the bead.
[0101] In an embodiment, the method further comprises amplifying
the nucleic acids released after contacting with a Proteinase K,
but prior to sequencing.
[0102] Also provided is a method of multiplex detecting a
protein-nucleic acid interaction, in a sample, for up to four
distinct proteins, proteins A, B, C and D respectively, the method
comprising:
[0103] contacting the sample with a (i) a first agent attached to a
surface of a magnetic bead that is not labeled with a first primary
optically-active label, and (ii) a second primary agent attached to
a surface of a magnetic bead that is labeled with a first primary
optically-active label, and (iii) a third primary agent attached to
a surface of a non-magnetic bead that is not labeled with a second
primary optically-active label, and (iv) a fourth primary agent
attached to the surface of a non-magnetic bead that is labeled with
a second primary optically-active label,
wherein the first, second, third and fourth primary agents are
different agents each capable of capturing the distinct first
proteins A, B, C and D, respectively, under conditions which permit
capturing to the primary agents a first protein-nucleic acid
complex from the sample;
[0104] contacting captured first protein-nucleic acid complex(es)
with a plurality of secondary agents each specific for a distinct
nucleic acid, and each labeled with a separate secondary
optically-active label wherein the secondary optically-active
labels are not the same as the primary optically-active labels of
the primary agents and are each distinct from the secondary
optically-active label of every other optically-active labeled
secondary agent;
[0105] recovering magnetic beads complexes from the sample by
applying a magnetic field;
[0106] recovering non-magnetic bead complexes from the sample based
on a non-magnetic physical property of the non-magnetic beads;
[0107] passing the recovered magnetic bead complexes through a flow
cytometer or optical plate reader and quantifying the optical
signal emitted therefrom;
[0108] passing the recovered non-magnetic bead complexes through a
flow cytometer or optical plate reader and quantifying the optical
signal emitted therefrom;
[0109] quantifying the optical signal(s) of the recovered magnetic
bead complexes; and
[0110] quantifying the optical signal(s) of the recovered
non-magnetic bead complexes;
[0111] wherein the presence on a magnetic bead complex of only a
secondary optically-active label indicates the interaction between
the first protein A and a nucleic acid corresponding to the
secondary optically-active labeled secondary agent,
and wherein the presence on a magnetic bead complex of both (i) a
first primary optically-active label and (ii) a secondary
optically-active label indicates the interaction of the first
protein B and a nucleic acid corresponding to the secondary
optically-active labeled secondary agent, and wherein the presence
on a non-magnetic bead complex of only a secondary optically-active
label indicates the interaction of the first protein C and a
nucleic acid corresponding to the secondary optically-active
secondary labeled agent, and wherein the presence on a non-magnetic
bead complex of both (i) a second primary optically-active label
and (ii) a secondary optically-active label indicates the
interaction of the first protein D and a nucleic acid corresponding
to the secondary optically-active labeled secondary agent.
[0112] In an embodiment of the methods, the presence on a bead
complex of a first primary optically-active label and/or a
secondary optically-active label is determined by quantifying the
optical signal thereof.
[0113] In an embodiment of the methods, the optical signal is
collected with one or more photomultipliers. In an embodiment of
the methods the recovered bead complexes are passed through a flow
cytometer. In an embodiment of the methods the recovered bead
complexes are passed through an optical plate reader.
[0114] In an embodiment of the methods, the methods further
comprise quantifying the optical signal(s) detected and comparing
the quantified amount against a control amount or control curve so
as to thereby quantify the amount of first protein-second protein
interaction on the bead or the amount of protein-nucleic acid
interaction on the bead, as relevant. Such a method may be termed
"digital cell Western" or "DCW."
[0115] In an embodiment of the methods, each primary agent
comprises an antibody or comprises an antigen-binding fragment of
an antibody.
[0116] In an embodiment of the methods, each secondary agent
comprises an antibody or comprises an antigen-binding fragment of
an antibody. In an embodiment of the methods, the antibodies are
monoclonal antibodies. In an embodiment of the methods, the
antibody fragments are F(ab')2 fragments, Fab' fragments or
ScFvs.
[0117] In an embodiment of the methods, the primary agents
comprises an antibody or comprise an antigen-binding fragment of an
antibody, and wherein the secondary agents are oligonucleotides
that hybridize with a specific sequence of a nucleic acid.
[0118] In an embodiment of the methods, the sample is a cell or
tissue lysate, or any biological lysates. In an embodiment of the
methods, the sample is a cell, and the cell is fixed and
permeabilized so as to permit primary and second agent entry into
the cell prior to performing the method.
[0119] In an embodiment of the methods, the first protein-second
protein complex comprises a protein of that is a product of a gene
variant.
[0120] In an embodiment of the methods, the first proteins A, B, C
and D are distinct variant forms of a single protein.
[0121] In an embodiment of the methods, the sample contains at
least one first distinct protein-second distinct protein
interaction.
[0122] In an embodiment of the methods, a bead complex comprises
the bead having a primary agent attached thereto, wherein the
primary agent is attached to a distinct first protein and the
distinct first protein is interacting (for example, bound to) a
distinct second protein which has bound a labeled secondary agent.
In an embodiment of the methods, a first protein-second protein
complex is an association of the first protein and second protein
(for example, by way of binding to each other).
[0123] In an embodiment, the interaction is an intermolecular
interaction, occurring by intermolecular forces, such as ionic
bonds, hydrogen bonds or van der Waals forces.
[0124] A "distinct" protein is one that has a sequence which is
non-identical to every other recited "distinct" protein. The
distinct proteins referred to herein are distinct in that they have
different amino acid sequences. The distinct proteins can be
variants, or can be completely different proteins. "Proteins A, B,
C and D," or grammatical variations thereof, as referred to herein
are not actual protein names, but merely identifiers to distinguish
up to four different proteins.
[0125] In an embodiment the term "variant," for example, of a gene
or a protein, means one having 97%, 98% or 99% or greater (but not
100%) sequence identify with the gene or protein, respectively,
that the recited gene or protein is a variant of. In an embodiment
the term "variant," for example, of a gene or a protein, means one
having 99% or greater (but not 100%) sequence identify with the
gene or protein, respectively, that the recited gene or protein is
a variant of.
[0126] In an embodiment of the methods, forward scatter (FSC)
and/or side scatter (SSC) are adjusted with a control un-complexed
bead population prior to initiating the method so as to permit
complexed beads to be detected.
[0127] In an embodiment of the methods, the ratio of primary agent
to primary optically-active labels on primary agents so-labeled is
1:1. In an embodiment of the methods, the ratio of secondary agent
to secondary optically-active labels on secondary agents so-labeled
is 1:1.
[0128] In an embodiment of the methods, the magnetic beads are
surface enhanced (e.g.: epoxy-coated) magnetic beads. In an
embodiment of the methods, the non-magnetic beads are carboxyl
modified beads.
[0129] In an embodiment of the methods, the agents are attached to
the beads by covalent binding.
[0130] In an embodiment of the methods, optically-active labels are
chosen from the group of fluorophores and nanocrystals.
[0131] In an embodiment of the methods, the sample is a lysate and
magnetic beads are separated from the lysate using a magnetic
field.
[0132] In an embodiment of the methods, the optical signal is
quantified by first exposing the complexed beads to one or more
excitation light sources, such as, in a non-limiting embodiment, a
laser. In an embodiment of the methods, the optical signal is
quantified by first exposing the complexed beads to one or more
lasers.
[0133] In an embodiment of the methods, the sample is contacted
with the first agent(s) and second agent(s) under conditions which
permit capturing to the primary agents a first protein-second
protein complex from the sample.
[0134] In an embodiment of the methods, the sample is a lysate and
the non-magnetic beads are separated from the lysate by
centrifugation.
[0135] In an embodiment of the methods, the cell or tissue lysate
is from primary isolated cells, lymphoblasts, fibroblasts, cancer
cells, a cell line, transfected cells, tissue or blood.
[0136] In an embodiment of the methods, quantitatively measured
optical activity of labeled agents bound to the complex is
converted into a relative or an absolute quantitation number of
co-binding molecules in each complex.
[0137] In an embodiment of the methods, forward scatter amplitude
gain and side scatter voltage on a flow cytometer are set to
register populations of bead events to on scale, followed by
applying an inclusion gate where selected linear populations of
beads form collective clusters containing interrogation targets can
be analyzed in their entirety by flow cytometry.
[0138] Also provided is a kit for detecting changes in protein
expression in cells and for analysis of gene variants, the kit
comprising:
magnetic beads for immunoprecipitation, non-magnetic beads for
immunoprecipitation, a lysis formulation, one or more Proteinase K
inhibitors, one or more phosphatase inhibitors, a coupling buffer,
nucleic acids recovery elution buffer, one or more functional
variant assay (FVA) buffers, a Western loading buffer, one or more
optically active labels, and instructions for use of the kit.
[0139] In an embodiment, the kit further comprises one or more
of:
a nucleic acid recovery buffer, a proteinase inhibitor, a
fix-permeabilization buffer, and one or more primary agents for
capturing protein-protein complexes or protein-nucleic acid
complexes.
[0140] In an embodiment, the optically active labels are
nanoparticles and/or fluorescent dyes.
[0141] In an embodiment, primary agents are attached to the beads.
In an embodiment, a portion and/or subset of the beads are labeled
with an optically active agent.
[0142] Also provided is a method of detecting and analyzing a gene
variant based on a protein-protein interaction, the method
comprising:
[0143] attaching a first primary agent to the surface of magnetic
beads that are not labeled with an optically active label and
attaching a second primary agent to the surface of magnetic beads
that are labeled with an optically active label;
[0144] attaching a third primary agent to the surface of
non-magnetic beads that are not labeled with an optically active
label and attaching a fourth primary agent to the surface of
non-magnetic beads that are labeled with an optically active label,
wherein the first, second, third and fourth primary agents are
different agents and wherein the first, second, third and fourth
primary agents are each capable of capturing a distinct protein
complex from a cell or tissue lysate;
[0145] capturing to the primary agents a protein complex from a
cell or tissue lysate, where the protein complex comprises a
protein of interest that is a product of a gene or a gene variant
and where the protein of interest forms part of a complex with
another protein;
[0146] probing the protein-protein complex with one or more
optically active secondary agents specific for a member of the
complex; wherein the same one or more optically active labels can
be used to label secondary agents on any of i) the magnetic beads
that are not labeled with an optically active label, ii) the
magnetic beads that are labeled with an optically active label,
iii) the non-magnetic beads that are not labeled with an optically
active label, and iv) the non-magnetic beads that are labeled with
an optically active label;
[0147] separating protein-magnetic bead complexes from the lysate
based on magnetic properties of the magnetic beads;
[0148] separating protein-non-magnetic bead complexes from the
lysate based on a physical property of the non-magnetic beads;
and
[0149] measuring optical activity of optically active-labeled
agents on the protein-bead complexes,
wherein the absence or presence of the optically active label on
the magnetic beads is used to distinguish optically active protein
complexes captured by the first and second primary agents,
respectively, and wherein the absence or presence of the optically
active label on the non-magnetic beads is used to distinguish
optically active protein complexes captured by the third and fourth
primary agents, respectively.
[0150] Also provided is a method of detecting and/or analyzing a
gene variant based on a protein-nucleic acid interaction, the
method comprising:
[0151] attaching a first primary agent to the surface of magnetic
beads that are not labeled with an optically active label and
attaching a second primary agent to the surface of magnetic beads
that are labeled with an optically active label;
[0152] attaching a third primary agent to the surface of
non-magnetic beads that are not labeled with an optically active
label and attaching a fourth primary agent to the surface of
non-magnetic beads that are labeled with an optically active label,
wherein the first, second, third and fourth primary agents are
different agents and wherein the first, second, third and fourth
primary agents are each capable of capturing a distinct
protein-nucleic acid complex from a cell or tissue lysate;
[0153] capturing to the primary agents a protein-nucleic acid
complex from a cell or tissue lysate, where the protein-nucleic
acid complex comprises a gene or a gene variant nucleic acid
sequence;
[0154] separating protein-nucleic acid-magnetic bead complexes from
the lysate based on magnetic properties of the magnetic beads;
[0155] separating protein-nucleic acid-non-magnetic beads complexes
from the lysate based on a physical property of the non-magnetic
beads;
[0156] digesting proteins on the protein-nucleic acid bead
complexes to release nucleic acids; and
[0157] amplifying the released nucleic acids;
wherein the absence or presence of the optically active label on
the magnetic beads is used to distinguish optically active
protein-nucleic complexes captured by the first and second primary
agents, respectively, and wherein the absence or presence of the
optically active label on the non-magnetic beads is used to
distinguish optically active protein-nucleic acid complexes
captured by the third and fourth primary agents, respectively.
[0158] The invention provides a method of detecting and/or
analyzing a gene variant based on changes of protein-protein
interactions, the method comprising:
[0159] attaching a first primary agent to the surface of magnetic
beads that are not labeled with an optically active label and
attaching a second primary agent to the surface of magnetic beads
that are labeled with an optically active label;
[0160] attaching a third primary agent to the surface of
non-magnetic beads that are not labeled with an optically active
label and attaching a fourth primary agent to the surface of
non-magnetic beads that are labeled with an optically active label,
wherein the first, second, third and fourth primary agents are
different agents and wherein the first, second, third and fourth
primary agents are each capable of capturing a distinct protein
complex from a cell or tissue lysate;
[0161] capturing to the primary agents a protein complex from a
cell or tissue lysate, where the protein complex comprises one or
more proteins of interest, where the protein of interest is a
product of a gene or a gene variant and where the protein of
interest forms a complex with another protein;
[0162] probing the protein-protein complex with one or more
optically active labeled secondary agents specific for a member of
the complex; wherein the same one or more optically active labels
can be used to label secondary agents on any of i) the magnetic
beads that are not labeled with an optically active label, ii) the
magnetic beads that are labeled with an optically active label,
iii) the non-magnetic beads that are not labeled with an optically
active label, and iv) the non-magnetic beads that are labeled with
an optically active label;
[0163] separating protein-magnetic bead complexes from the lysate
based on magnetic properties of the magnetic beads;
[0164] separating protein-non-magnetic bead complexes from the
lysate based on a physical property of the non-magnetic beads;
and
[0165] measuring optical activity of optically active-labeled
agents probed on the protein-bead complexes,
[0166] wherein the absence or presence of the optically active
label on the magnetic beads is used to distinguish optically active
protein complexes captured by the first and second primary agents,
respectively, and
[0167] wherein the absence or presence of the optically active
label on the non-magnetic beads is used to distinguish optically
active protein complexes captured by the third and fourth primary
agents, respectively.
[0168] The invention also provides a method of detecting and/or
analyzing a gene variant based on changes of protein-nucleic acid
interactions, the method comprising:
[0169] attaching a first primary agent to the surface of magnetic
beads that are not labeled with an optically active label and
attaching a second primary agent to the surface of magnetic beads
that are labeled with an optically active label;
[0170] attaching a third primary agent to the surface of
non-magnetic beads that are not labeled with an optically active
label and attaching a fourth primary agent to the surface of
non-magnetic beads that are labeled with an optically active label,
wherein the first, second, third and fourth primary agents are
different agents and wherein the first, second, third and fourth
primary agents are each capable of capturing a distinct
protein-nucleic acid complex from a cell or tissue lysate;
[0171] capturing to the primary agents a protein-nucleic acid
complex from a cell or tissue lysate, where the protein-nucleic
acid complex comprises a gene or a gene variant nucleic acid
sequence;
[0172] separating protein-nucleic acid-magnetic bead complexes from
the lysate based on magnetic properties of the magnetic beads;
[0173] separating protein-nucleic acid-non-magnetic bead complexes
from the lysate based on a physical property of the non-magnetic
beads;
[0174] digesting proteins on the protein-nucleic acid complexes to
release nucleic acids; and
[0175] amplifying the released nucleic acids;
[0176] wherein the absence or presence of the optically active
label on the magnetic beads is used to distinguish optically active
protein-nucleic complexes captured by the first and second primary
agents, respectively, and
[0177] wherein the absence or presence of the optically active
label on the non-magnetic beads is used to distinguish optically
active protein-nucleic acid complexes captured by the third and
fourth primary agents, respectively.
[0178] In any of the methods disclosed herein, a washing step can
be performed between contacting and recovering steps in order to
remove unwanted or unbound materials.
[0179] In any of the methods or kits disclosed herein, the magnetic
beads can be, for example but not limited to, epoxy-coated magnetic
beads. In any of the kits or methods disclosed herein, the
non-magnetic beads can be, for example but not limited to, carboxyl
modified beads. Examples of beads that can be used he methods and
kits disclosed herein include, but are not limited to,
Dynabeads.RTM. M-270, Dynabeads.RTM. M-450, Carboxyl Modified Latex
Beads and Dynabeads ClinExVivo Epoxy from Invitrogen Corporation,
Carlsbad Calif.
[0180] Additional agents can be attached the surface of the beads,
and protein-protein and protein-nucleic acid complexes, to increase
the complexity of the functional assay.
[0181] In any of the methods or kits disclosed herein, the agents
can be one or more of antibodies, monoclonal antibodies, polyclonal
antibodies, antibody fragments, F(ab').sub.2 fragments, Fab'
fragments, peptides, nucleotides, peptide nucleic acids, and small
biological and/or chemical compounds. The small compound can have a
molecular weight of, for example but not limited to, 2,000 daltons
or less, e.g., 1,000-2,000 daltons.
[0182] In any of the methods or kits disclosed herein, agents can
be attached to the beads, for example but not limited to by
chemical binding, such as, e.g., covalent binding.
[0183] In any of the methods or kits disclosed herein, beads can be
labeled with an optically active label by, for example, using an
optically active primary agent. Alternatively, or in addition,
beads can be labeled with an optically active label by using an
optically active agent that is different than the primary agent
used to capture protein complexes or protein-nucleic acid
complexes.
[0184] In any of the methods or kits disclosed herein, the
optically active label can be, for example but not limited to, a
fluorescent label and/or a nanocrystal (e.g., QDot.RTM.).
[0185] An optically active label having the same unique wavelength
(for example, color green) can be used to label secondary agents on
any of i) the magnetic beads that are not labeled with an optically
active label, ii) the magnetic beads that are labeled with an
optically active label, iii) the non-magnetic beads that are not
labeled with an optically active label, and iv) the non-magnetic
beads that are labeled with an optically active label. This allows
for multiplexing of labels to identify distinct protein-protein
and/or protein-nucleic acid complexes.
[0186] In any of the methods disclosed herein, the magnetic beads
can be separated from the lysate using a magnetic field. In methods
disclosed herein, the non-magnetic beads can be separated from the
lysate their physical properties, for example but not limited to
centrifugation. Alternatively, after the magnetic beads are removed
from the lysate, non-magnetic beads can be separated from the
lysate by binding (covalent or non-covalent) the non-magnetic beads
to secondary magnetic beads and separating the bound
non-magnetic/magnetic bead complex using a magnetic field.
[0187] In methods disclosed herein, quantitatively measured optical
activity of optically active agents bound to the complex can be
converted into an absolute or estimated relative number of
co-binding molecules in the complex. Optically active beads can be
counted by optical readers, for example but not limited to a flow
cytometer and/or plate reader.
[0188] In methods disclosed herein, the methods can comprise
techniques of selecting subpopulation of beads with analyte complex
by means of sorting for particular targets of interest based on
optical properties for further analysis and/or downstream
applications, for example but not limited to purification of the
captured analytes.
[0189] In methods disclosed herein, forward scatter amplitude gain
and side scatter voltage on a flow cytometer can be set to register
populations of bead events to on scale, followed by applying an
inclusion gate where linear selected populations of beads are
analyzed so that complex populations of the collective clusters
containing interrogation targets can be analyzed in their entirety
by flow cytometry.
[0190] In methods disclosed herein, captured/purified nucleic acids
can be amplified using any conventional method, e.g., sequencing,
polymerase chain reaction, multiplex ligation assay, etc.
[0191] In methods disclosed herein, the cell or tissue lysate can
be from, e.g., primary isolated cells, such as, lymphoblasts,
fibroblasts, normal or diseased tissues, cancer cells or any cell
lines not limited to transfected cells and from tissues or
blood.
[0192] With any of the methods or kits disclosed herein, a
wild-type gene can be compared with a gene variant with or without
transgene overexpression (exogeneous).
[0193] The invention provides a kit for detecting changes in
protein expression in cells and for analysis of gene variants, the
kit comprising:
[0194] magnetic beads for immunoprecipitation,
[0195] non-magnetic beads for immunoprecipitation,
[0196] a lysis formulation,
[0197] one or more Proteinase K inhibitors,
[0198] one or more phosphatase inhibitors,
[0199] a coupling buffer,
[0200] nucleic acids recovery elution buffer,
[0201] one or more (e.g., a set of) functional variant assay (FVA)
buffers,
[0202] a Western loading buffer,
[0203] one or more optically active labels, and
[0204] instructions for use of the kit.
The optically active labels can be, for example but not limited to,
nanoparticles and/or fluorescent dyes.
[0205] The kit can also include, for example, one or more of a
nucleic acid recovery buffer (CHIP digest buffer), a protease
inhibitor, a fix-permealization buffer, and one or more primary
agents for capturing protein-protein complexes or protein-nucleic
acid complexes.
[0206] The kit can include one or more primary agents for capturing
protein-protein complexes or protein-nucleic acid complexes. The
primary agents can be attached to the beads. A portion and/or
subset f the beads can be labeled with an optically active agent.
The kit can include, for example, one or more optically active
agents for binding to a protein-protein complex or to a
protein-nucleic acid complex.
[0207] The kit and methods disclosed herein allow techniques of
selecting subpopulation of beads with analyte complex by means of
sorting for particular targets of interest for further analysis
and/or downstream applications, for example but not limited to
purification of the captured analytes.
[0208] Examples of phosphatase inhibitors than can be used include,
but are not limited to, 10.times. sodium orthovanadate stock and
10.times. sodium fluoride stock. Examples of Proteinase K
inhibitors than can be used include, but are not limited to,
10.times. Super Proteinase K inhibitor cocktail. For example,
dissolve Proteinase K inhibitor cocktail (Sigma, cat. no. P 2714)
in 900 .mu.L distilled H.sub.2O. It also contains other inhibitors,
for example but are not limited to AEBSF
(4-(2-aminoethyl)benzenesulfonyl fluoride, Sigma, cat. no. A8456),
bestatin, aprotinin, Ethylenediaminetetraacetic acid (EDTA), E-64,
and leupeptin. Examples of detergents that can be used include, but
are not limited to, nonionic detergent: e.g. Triton X-100, NP-40,
and digitonin.
[0209] The instructions for use of the kit can include any of the
instructions set forth within the present application.
[0210] The invention provides kits for the following:
1. Identification of the Protein-Protein Interactions;
[0211] 1.1. The kit includes a bar-coded bead system, for example
but not limited to epoxy-modified Dynabeads and CML beads, buffers,
and recommended optically active agents, for example but not
limited to fluorescent dyes (one vial for each conjugate dyes for
attaching to an agent, for example but not limited to target
antibodies). A complement of buffer system for all steps from
fixation, permeabilization and staining of cells, lysis of cells or
tissues, protein, FVA washes and flow analysis.
2. Protein-Nucleic Acid Interactions;
[0211] [0212] 2.1. Gentle elution buffer for active protein-nucleic
acid complex purification, and sample recovery after FVA analysis
by sorting. The kit includes a protein digestion chemistry in the
form of, for example but not limited to, DNase and RNAse-free
proteinase K, Upon performing FVA or Flow sorting, population of
beads can be precipitated by applying magnetic field, and by
physical separation of the second population of beads with another
complex. No wash needed. A kit digestion buffer mix is added to the
barcoded bead systems for 2 hour or overnight digest to the beads.
DNA elution and purification at neutral pH to enrich captured
nucleic acids for applications of MPS followed after CHIP, allelic
discrimination assays, quantitative PCR expression analysis, RNA
IP, RNA CHIP and CHIPs PCR assays.
3. Cell-Based Protein Expression;
[0212] [0213] 3.1. A digital cell western system (DCW) to fix and
permeabilize cells for staining of internal cellular expression of
analytes is designed in the kit. This replaces the need to perform
separate Western blot prior to IP, or standard westerns.
4. Protein Modifications;
[0213] [0214] 4.1. With the available of antibodies for modified
proteins (phosphorylation, acetylation, prenylation,
ubiquitination), and other antibodies or agents that can detect
modifications of analyte, the kit is design to incorporate these
agents as part of the unified assay read out.
5. Concurrent Immunoprecipitation (IP)-Western;
[0214] [0215] 5.1. The recovered beads after FVA analysis can be
treated with the kit Western elution buffer containing Sodium
dodecyl sulfate (SDS) and dye designed for downstream traditional
Western blot. This reduces technical variations when comparing
experimental readout data from the same sample.
6. Generation of Reference Controls Using the FVA Kit Conjugated
Antibodies;
[0215] [0216] 6.1. The conjugation buffer is adapted to perform
conjugation of the freshly made optically active, for example but
not limited to fluorescently labeled, antibodies to the beads
rapidly. These are the antibodies that will be used to detect the
FVA analytes. This increases consistency as the same host, and
recognition epitope as in the detection antibodies are used.
[0217] The invention provides methods that include:
1. Methods to Identify the Protein-Protein Interactions;
[0218] 1.1. Bait antibody is conjugated on to the surface of the
epoxy modified beads, a set of optically active multi-analyte
antibodies can be used to detect the bound proteins using methods
of FVA. This allows identification of proximal binding targets
within one complex. FVA allows quantitative measurement of up to
various targets simultaneously. With the use of FVA, selection of
particular variant interactions can be selected by gating
inclusion/exclusion sorter, and be eluted for further assays and
other analysis that is otherwise not possible by conventional
means. The kit includes steps to use these same analyte agents,
such as for example antibodies, to generate optical standards that
can be used to accurately establish the threshold of detection
based on its optical standard intensities.
2. Methods to Identify Protein-Nucleic Acid Interactions;
[0218] [0219] 2.1. A simplified workflow that includes nucleic acid
enrichment after FVA by elution of protein-nucleic acid complex,
digest, release of nucleic acids and enrichment methods, for
example but not limited to ligation of adaptamers to the fragments
of captured nucleic acids to perform digital emulsion or standard
PCR steps to assess the bound regions to the target sequences and
their variants using optical scanners, for example sequencer, flow
cytometer or plate reader. The target sequence and its variant
forms (single nucleotide polymorphisms, insertions and deletions,
and structural rearrangements) can be measured by using optically
active probes annealed to the amplified targets on the beads. In
addition, the kit allows novel methods to identify binding sites of
the RNA with the interacting proteins in its native form, adapted
to a high throughput and high content analysis format.
3. Cell-Based Protein Expression (DCW);
[0219] [0220] 3.1. Prior to lysing of the cells for FVA, a quick
assessment of the expression levels of the analyte can be performed
by sampling using DCW to fix, permealize, and immunostain the
cells. Multiple protein targets can be measured simultaneously.
Each detention data point is considered to be one cell western, and
aggregate of cells generates a digital value of the cumulative data
points, as digital cell western.
4. Protein Modifications;
[0220] [0221] 4.1. DCW methodology is optimized to incorporate
available analyte antibodies to detect protein modifications, where
the antibodies for specific post translational modification can be
conjugated with the optically active agent.
5. Concurrent Immunoprecipitate (IP)-Western;
[0221] [0222] 5.1. Beads that are retained after FVA analysis can
be placed into denaturing western loading buffer in the kit to
perform standard IP-western blot.
6. Generation of Reference Controls Using the Optically Active
Conjugated Antibodies;
[0222] [0223] 6.1. In traditional protocols, prior to performing
flow cytometry analysis, a set of commercial bead-based reference
dyes is used to create fluorescence standards; drawbacks are
commonly acknowledged that the host epitope of antibodies for the
analyte is greatly different from the references. The FVA reference
control methodology resolve this issue by using the same conjugated
antibodies as calibration controls by conjugating them on to the
bead systems.
[0224] The interaction of the protein of interest ("bait") or
variant protein with its associated targets, and multiple gene
variant complexes can be examined simultaneously. This has the
effect of looking not only at a major effect of a variant in the
bait gene(s), but also at the epistatic interactions of a variant
in second gene(s) with the bait products of other genes in the
complex.
[0225] By altering the gating and an inclusion principle of
counting a population of subsets of beads previously missed, the
present invention greatly improves sensitivity by at least 10-fold.
By improving the sensitivity, the amount of starting material can
be reduced significantly and by using a bar-coded bead system, the
binding time can be reduced dramatically from 24 hours (overnight)
to 5 hours for multigene functional assay.
[0226] By gating and sorting for analytes of interests, the present
invention allows for selection and enrichment of target proteins or
nucleic acids that would simply not be possible using current
technologies.
[0227] The kit provides all the steps and chemical reagents
necessary to perform a DCW assay, with addition of one initial
startup step, i.e., permeabilize the cells prior to the FVAstandard
protocol.
[0228] As used herein, one form of "bar coding" means to identify
different protein-protein complexes or different protein-nucleic
acid complexes on beads with different optically active labels
("vertical bar coding"). Another form of bar-coding means to
identify different analytes on the magnetic beads and/or
non-magnetic beads using optically active labels ("horizontal bar
coding"). For example, but not limited to, for 7 different
fluorescent labels, one label can be used to identify a portion
and/or subset of magnetic or non-magnetic beads and the remaining 6
labels can be used to label different protein-protein complexes or
different protein-nucleic acid complexes attached to the beads.
[0229] Examples of apparatus that can be used with the disclosed
methods include, but are not limited to, a 7-color flow laser
machine or a 13-color flow laser.
[0230] The methods disclosed herein allow unprecedented speed to
study effects of mutations and offers high sensitivity to detect
mutant protein activities, such as changes in protein-protein
interactions, modifications and mutant protein co-localization into
the nucleus. Traditionally, a transgene is needed for each
interrogated mutant, whereas the present method uses cells isolated
from actual subject bearing the mutation, hence there is no need to
create any transgene prior to the FVA assay. In an embodiment of
the methods, no transgene is inserted into the cells of the sample.
Additionally, conventional methods struggle to detect endogenous
protein accurately, but the increased sensitivity of the present
methods allow accurate detection of the mutant protein and of its
activities. This changes the paradigm of having to genetically
engineer mutant cells to study its biology, to simply using the
isolated subject cells for various assays described. In an
embodiment of the methods, no the cells of the sample have not been
previously genetically engineered. The methods herein allow
effective cost reduction for large-scaled studies of mutation.
[0231] This invention will be better understood from the
Experimental Details that follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims that follow thereafter.
EXPERIMENTAL DETAILS
Example A
Methods
Introduction
[0232] The dynamic range of Co-IP Western blots can be improved by
flow cytometry, a direct counting function (13). Using this method,
immunoprecipitating antibodies, known as `bait`, are coupled
covalently to polystyrene beads whose low autofluorescence is
suitable for flow cytometry detection. Once coupled, the antibodies
on beads will immunoprecipitate a specific protein complex in cell
lysates. These complexes can then be probed with a panel of
fluorescently tagged secondary antibodies to quantify the binding
of the interacting partners by quantitatively count the bound beads
on a flow cytometer at high speed. Here, this method has been
applied to probe a clinical case study using wild-type B
lymphoblastoid cell lines or those derived from patients with
MAP3K1 mutations who have 46,XY gonadal dysgenesis (14).
[0233] The present studies show altered binding of interacting
proteins that could influence downstream signaling in testis or
ovarian-determining pathways. Because B cell lines express a large
repertoire of protein complexes and can be derived from patients
with presumed genetic disorders in a non-invasive manner, they
represent a readily available resource for screening variants of
uncertain phenotypic significance.
Materials and Methods
[0234] Cell culture and reagents. Three MAP3K1 mutation-bearing
Epstein-Barr virus B-lymphoblastoid cells lines (c.634-8A,
p.Leu189Arg, and p.Leu189Pro) from individuals with 46,XY complete
gonadal dysgenesis and a wild-type cell line were derived, as
described previously (14). These cell lines were grown in RPMI
medium (Invitrogen A2780), supplemented with 15% (v/v) FBS Defined
Grade, 50 units/ml penicillin, and 50 g/ml streptomycin at
37.degree. C. with 5% CO.sub.2. The antibodies included MAP3K1
rabbit polyclonal antibody (Lifetech #51-340), RHOA rabbit
polyclonal antibody (Abcam #ab66124), MAP3K4 mouse monoclonal
(Abcam #55669), IRDye 800CW Goat anti-Mouse (Licor #926-32210), and
IRDye 680LT Goat anti-Rabbit IgG (Licor #926-68021). Western
blotting analysis was performed as described previously (14).
[0235] FVA on Cultured Lymphoblastoid Cells.
[0236] FVA was performed with the following modifications optimized
for lymphoblastoid cell lysates. The bait antibody was conjugated
onto epoxy modified beads at concentrations of 30 .mu.g of antibody
per mg of 5 micron epoxy beads, conjugation is achieved using the
coupling buffers in the kit. The Proteinase K and phosphatase
inhibitors at 2 or 4.times. concentration were added to the cell
lysates during lysis. Antibody conjugated epoxy beads were added to
the lysates to incubate for 5 hours or overnight at 4.degree. C.
Alexa 488 and 647 dyes were coupled to the secondary antibodies
using labeling kits (Lifetech #A20181 and A20186, respectively).
The secondary antibodies were added to the lysate after 2 washes of
the previous incubation, and were incubated at room temperature
(20.degree. C.) for 30 minutes with 2 additional washes following
to remove unbound antibodies. Flow cytometry was performed on a BD
FacsCantoll with 96-well, high-throughput capabilities. The
counting of bead fluorescence occurrences, `events`, was set at
10,000-25,000 gated events setting from the previously published
2500 events (13). Note that `gating` refers to the combination of
fluorescence and scatter values that are counted.
Results
[0237] The intrinsic fluorescence of the kit beads was low and did
not interfere with subsequent analysis of binding fluorochromes.
The fluorescence of the Alexa 488 and 647 dyes on
antibody-conjugated epoxy beads was used, formulated using the kit
coupling buffers (see protocol step 2 in "Preparation before
starting") as reference control for calibrating the flow cytometer
gate sensor to exclude background noise and to gauge dye detection
and recognition fidelity. Positive controls from binding of either
MAP3K1 or RHOA complexes to the beads could, in turn, be recognized
by the labeled MAP3K1 and RHOA antibodies--demonstrating the
specificity of these IP pull-down reactions. Western blot analysis
was performed to confirmed that the expression of wild-type and
mutant MAP3K1 proteins and that the input amounts of MAP3K1 prior
to IP were consistent (FIGS. 4C and 6A). Previously, Western blot
analysis showed that the input amounts of RHOA prior to IP were
consistent (14). The IP Western from the MAP3K1 pull-down of
MAP3K4, and the RHOA pulldown of MAP3K1 in the aggregate showed
dramatic increases in binding among mutant samples as quantified by
densitometry analysis using Licor Software 3.0 (FIGS. 6B and 6C).
When applied to the analysis of the patient and control samples to
measure protein interactions, the binding of MAP3K4 was shown by
FVA to be increased 3-fold in these cell lines that contain any of
three endogenous mutant MAP3K1 genes (C.654-8A, p.Leu189Pro and
p.Leu189Arg) compared to wild-type cell lines (FIG. 4). The results
observed with FVA were confirmed by conventional IP Western blots
of samples eluted from the epoxy beads, although the binding
appeared to be increased 2-fold on average among all mutants,
similar results were observed in previous traditional IP Western.
Accordingly, it was confirmed that the increases observed were not
the result of unequal loading nor increased expression of MAP3K1,
as illustrated by actin Western as loading control and MAP3K1 input
prior to IP (FIG. 6B).
[0238] These methods were applied to the reverse
immunoprecipitation method in which the `bait` was RHOA, a
MAP3K1-interacting protein to show consistency, and binding of
wild-type or mutant MAP3K1 was measured by flow cytometry (FIG. 5).
In this case, the binding of the mutant MAP3K1 proteins was
increased 2 to 4-fold in cells containing mutant compared to
wild-type MAP3K1. The results observed with FVA were confirmed
consistent, and by conventional immunoprecipitation Western blots
of samples eluted from the epoxy beads, notably the binding from
all mutant cells appeared to be increased 2.5-fold compared to
wild-type (FIGS. 5C and 6C). This discordance has been accounted
for by the limited dynamic range of conventional Western blots
compared to the sensitivity of flow cytometry.
Discussion
[0239] DNA sequencing of candidate genes or whole exomes on a
diagnostic or investigational basis will yield a plethora of
variants whose potential phenotypic roles cannot be readily
demonstrated by prediction programs, SNP databases nor conventional
genetic analysis. Many variants may produce phenotypic changes in
the encoded proteins by affecting the quantity, post-translational
modification or protein interactions. The present studies establish
the application of the method of FVA to demonstrate that known
protein interactions are altered in the B lymphoblastoid cells of
patients with 46,XY gonadal dysgenesis arising from mutations in
the MAP3K1 gene. This method can be scaled readily to test multiple
interactions for many variants simultaneously from available
tissues as well as quantify the effects of variants on protein
accumulation and post-translational modification, thus functional
screening of gene variants for phenotypic effects is made possible
by this efficient and low-cost FVA.
[0240] Here, it was shown that the effects of missense and in-frame
splicing (with insertion of 2 amino acids) mutations in the amino
third of the MAP3K1 gene result in increased binding of MAP3K4.
This might arise through interactions with their shared binding
partner, AXIN1 (15-16). Both MAP kinases compete for AXIN binding,
albeit at different sites (15), and the presence of these MAP3K1
mutations may alleviate this competition. Unlike MAP3K1, MAP3K4 is
an essential testis determining gene. Homozygous loss of function
alleles in mouse Map3k4 lead to disrupted testis development in
mice from failure to support cord development (17), whereas
knockout of the mouse Map3k1 gene does not (18). Reduction of
MAP3K4 protein either from genetic knockout or from sequestration
in MAP3K1-MAP3K4 complexes may have functionally similar effects.
Increased binding of MAP3K4 to MAP3K1 complexes as shown by FVA may
affect downstream WNT targets through AXIN1. AXIN1, an inhibitor of
the WNT signaling pathway, interacts with .beta.-catenin to reduce
its protein abundance (16, 19). This results in an increase and/or
stabilization of .beta.-catenin, an effect that is known to cause
male-to-female sex reversal in the XY gonad--in part by reducing
SOX9 expression (20).
[0241] The present studies have also shown accurate detection of
changes of binding partners to MAP3K1 in all three mutant MAP3K1 by
FVA cases consistent with previous studies using traditional
methods but at a higher fidelity (21).
[0242] These approaches of FVA, either with or without
immunoprecipitation, can be applied to test other candidate gene
variant expressions and functional implications. In the process,
FVA can measure the general effects that a variant might have for a
protein and its regulation of downstream targets. High-content
measurements such as alteration of its expression and effects on
the expression and accumulation of other downstream proteins in the
cell, alteration of the post-translational modification of the
variant protein, such as phosphorylation, or alteration of the
variant and/or wild-type protein with its co-factors. Typically a
handful of variants are selected for biological assays by genetic
manipulation in cells or animals to show that a newly identified
variant is a mutation, i.e. has a phenotypic effect. Previously, it
has been shown that the effects on accumulation and
post-translational modification can be measured reliably by flow
cytometry in research and clinical applications (13, 27). The
improved methods of FVA and kit presented here have been tested on
human in-vitro studies using primary cells isolated from the
subjects, and compared to standard genetic manipulated human cell
lines to demonstrate for the use of FVA as high-throughput
screening for the effects of genetic variants on binding of
partners, at proteomic level (functional assay) that are readily
adaptable for single-tube, 96- or 384-well approach. Thus,
functional analysis of multiple variants and multiple binding
partners in a single experiment in one or two days can be performed
reliably and cost effectively. Moreover, comparison of previous
IP-Western densitometry results with this invention FVA shows a
greater sensitivity using only one-fifth the amount of starting
material compared to traditional methods. The multiple binding
partners can be tested in the same tube, because analytes using
optically active antibodies each with different emission
wavelengths can be measured simultaneously (27). Furthermore, the
method can test not only interactions with binding partner, but
also the quantification of the bound protein itself and its
post-translationally modified forms, such as the phosphorylation
status of the MAP kinase (28). The present methods and kits have
been extensively tested in several other clinical genetic studies
where B-lymphoblastoid cells isolated and immortalized from
patients were used; these type of blood cells are commonly use in
traditional genetic screening. As stated previously, the method was
also tested in Neuronal Teratocarcinoma 2 (NT2) cells, a standard
cell line routinely used in research laboratories to study gene
variant effects by genetic manipulations.
[0243] In conclusion, this method proves to have clinical
diagnostic value, a non-invasive, and cost effective test suitable
for virtually any variants that are identified by massive parallel
sequencing and genome-wide association studies (GWAS). In addition,
the methods can be used to survey and identify the genetic
heterogeneity of populations of cells. The methods can also be used
to investigate epistatic interactions between proteins, wherein one
protein is the distinct first protein of the method and the other
protein is a distinct second protein.
Example B
Kits
Introduction
[0244] Protein expression is deemed to be a gold standard for
measuring changes in gene activities. Many variants identified
through sequencing or mutant characterization may produce
phenotypic changes in the encoded proteins by affecting the
quantity, post-translational modification or protein interactions.
Indeed, the frequency of rare sequence variants is proving to be
far higher than previously thought. Complex protein interactions
play crucial roles in virtually all cellular processes.
Traditionally, such protein-protein interactions were studied via
co-immunoprecipitation. However, this method is laborious and are
only useful for small number of targets, require large amount of
biomaterial to start, not clinically sensitive and costly as each
target must be measured independently in separate Western blots.
Analysis of co-immunoprecipitation of protein complexes using FVA
provides a sensitive rapid method to measure multiples of these
interactions in their native state. This kit provides all the
essential and optimized reagents to perform the assay along with a
validated `bait` antibody. First, target bait antibodies are
covalently coupled to the bar-coded bead system in the kit. These
antibody-coupled beads are used as bait for protein lysates.
Finally, the pulled-down protein complexes on the beads are
separated based on their properties and probed with optically
active agent-labeled antibodies specific for interaction partners
and measurement of a quantitative fluorescence are converted into
quantity or numbers of co-binding molecules in a complex. FVA
represents a robust technique to assess native protein-protein
interactions rapidly with very small amount of biomaterials. This
kit includes, for example but not limited to, seven optically
active agents with minimal spectral overlap to measure multiple
targets simultaneously with a standard three-laser flow cytometer,
with methods that can be performed in just under one day, and
alternative protocol allows for a two-day assay if needed.
Absolute Quantitative Flow Cytometry
[0245] Optical activity data are often presented on a relative
scale with arbitrary units because it is inherently
semi-quantitative by traditional means. This kit allows both
measurements of semi and absolute quantification. This kit provides
a set of standard optically active reference beads created using
the same antibodies selected for each tests to generated standard
curve, where optical activity values correspond to known numbers of
molecules. Using this standard curve, on the same flow cytometer
settings, absolute quantitation can be measured by translating the
measured relative optical activity values from the FVA into numbers
of target molecule per IP bead.
Kit Components
[0246] Kit components can include:
Immunoprecipitate Beads (Store at 4.degree. C.)
[0247] Examples include, but are not limited to, polystyrene-epoxy
paramagnetic (Dynabeads) beads (5 .mu.M) in storage buffer,
carboxylate-modified polystyrene surface latex (CML) beads (5
.mu.m) in storage buffer, 10 ml of Flow grade neutral buffered salt
solution, pH 7.4. Examples of buffers include Phosphate Buffered
Saline, Phosphate Buffeted KCL, Trisaminomethane (Tris)-buffered
ammonium chloride.
Coupling Buffers (Store at 25.degree. C.)
[0248] Coupling buffers at pH range of 4-7 with use of agents, for
example but not limited to, 2-[N-morpholino]ethanesulfonic acid
(MES)) that allow covalent coupling of the agents on to the beads,
chelating agents, for example but not limited to EDTA, and
activating compounds, for example but not limited to
1-ethyl-3-(3-dimethylaminopropl) Carbodiimide HCl (EDAC) are added
as well.
Fix-Permealization Buffer (DCW Buffer) (Store at 4.degree. C.)
[0249] A kit for fix and permeabilizing cells comprising but not
limited to (a) an isotonic or hypertonic fixing agent at pH 4 to 7
containing, for example but not limited to an aliphatic aldehyde or
alcohol (glutaraldehyde, para- or formaldehyde, alcohol), which is
present in a concentration of at least 5%, (b) a permeabilizing
agent, for example but not limited to at pH 4 to 8 containing a
blocking agent (Bovine serum albumin, BSA) and selected group
consisting of Zwitterionic and the use of ionic detergent (e.g.,
Sodium dodecyl sulfate, SDS).
Inhibitor Cocktail (Store at -20.degree. C.)
Phosphatase Inhibitor Cocktail
[0250] A kit containing phosphatase inhibitor that inhibits
phosphatases. Examples of phosphatase inhibitors than can be used
include, but are not limited to, sodium orthovanadate and sodium
fluoride, and contain a preservative such as Sodium Azide.
Proteinase K Inhibitor Cocktail
[0251] A kit containing Proteinase K inhibitor that inhibits
Proteinase Ks including for example but not limited to
chymotrypsin, kallikrein, plasmin, thrombin, and trypsin. Examples
of inhibitors include, but are not limited to,
phenylmethanesulfonyl fluoride (PMSF) or
4-(2-Aminoethyl)benzenesulfonyl fluoride (AEBSF), and contain a
preservative such as Sodium Azide.
Universal Buffer (Blocking and Storage)
[0252] A buffer containing, for example but not limited to,
blocking agent (BSA), host serums (Rabbit, mouse, and/or goat
serum), preservative (Sodium Azide). Formulations include isotonic
and hypertonic saline, buffering agent and salts, examples include
but not limited to Phosphate buffer saline (PBS), Potassium
chloride (KCL), Monopotassium phosphate (KH.sub.2PO.sub.4), Sodium
chloride (NaCl), Disodium hydrogen phosphate (Na2HPO4) and
Tris.
After-IP Buffer
[0253] A buffer containing, for example but not limited to,
Proteinase K inhibitors (PMSF or AEBSF), preservative (Sodium
Azide), isotonic and hypertonic saline, buffering salts, examples
include but not limited to Sodium chloride (NaCl).
FVA Buffer
[0254] A buffer containing, for example but not limited to,
blocking agent (BSA), host serums (Rabbit, mouse, and/or goat
serum), preservative (Sodium Azide), isotonic and hypertonic
saline, buffering agent and salts, examples include but not limited
to Phosphate buffer saline (PBS), Potassium chloride (KCL) or
Sodium chloride (NaCl) and Tris.
Lysis Buffer
[0255] A buffer containing, for example but not limited to, a
buffering agent (Tris), Proteinase K inhibitors (PMSF or AEBSF),
phosphatase inhibitors (Sodium Orthovanadate, Sodium Fluoride), a
chelating agent (EDTA), contains hypertonic salt, examples include
but not limited to Potassium chloride (KCL) and/or Sodium chloride
(NaCl), non-ionic detergent (Digitonin) or ionic detergent
(SDS).
Elution Buffer (Store at 4.degree. C.)
[0256] A buffer containing, for example but not limited to,
blocking agent (BSA), Proteinase K inhibitors (PMSF or AEBSF),
phosphatase inhibitors (Sodium Orthovanadate, Sodium Fluoride), a
chelating agent (EDTA), a preservative (Sodium Azide), contains
hypertonic salt, examples include but not limited to Potassium
chloride (KCL) and/or Sodium chloride (NaCl), a buffering agent
(Trisaminomethane (Tris)), non-ionic detergent (Digitonin) or ionic
detergent (SDS, or Triton X-100).
Nuclear Enrichment Buffer (Store at 4.degree. C.)
[0257] To remove the cytoplasmic compartment of the cells with or
without fixation (DCW buffer), resuspend cell pellet in NP40 with
Tween 20 lysis buffer: 30 mM Tris/Hepes, pH 8.0, (0-50 mM) NaCl, 3
mM EDTA, 1.5 mM phenylmethylsulfonyl, fluoride (PMSF or equivalent
agent), 1% Tween 20 and 1% NP40. Leave on ice for 15 min with
pipetting for 5 times, then pellet the nuclei at 2000-6000 g for 3
min at +4.degree. C. Collect or decant the supernatant (cytoplasm
fraction) supplemented with 300 mM NaCl if needed for FVA analysis.
Resuspend the nuclear pellet in 100 ul of Universal buffer
containing host IgG and store 24 hours in 4.degree. C. prior to
assay, add 120 ul of methanol if long term storage is desired.
Western Loading Buffer
[0258] A buffer containing, for example but not limited to, ionic
detergent (SDS, or Triton X-100), a buffering agent (Tris),
coloring agent (Bromophenol blue), a hygroscopic simple polyol
compound (glycerol) and a small-molecule redox reagent such as
Cleland's reagent, example include but not limited to
Dithiothreitol (DTT) or dithioerythritol (DTE).
Nucleic Acids Recovery Elution Buffer Mix
[0259] A buffer containing, for example but not limited to, protein
digesting agent (proteinase K), a chelating agent (EDTA), isotonic
and hypertonic saline, detergent buffering agent and salts,
examples include but not limited to Phosphate buffer saline (PBS),
Potassium chloride (KCL), potassium phosphate (KHPO.sub.4), Calcium
chloride (CaCl), Glycine, Tris.
Protease Inhibitor Cocktail
[0260] A buffer containing, for example but not limited to,
proteinase inhibitors PMSF and Diisopropyl phosphorofluoridate
(DPF)) and a chelating agent (Ethylene glycol tetraacetic acid,
(EGTA)).
Preparation Before Starting
[0261] Coupling of Bait Antibodies to Beads.
[0262] Pipette 20 .mu.L (18.times.10.sup.6) Dynabeads into a 1.5-mL
microcentrifuge tube. Wash the beads 2 times in 700 .mu.L Buffer
C1, magnetize the tube and remove supernatant after each wash.
Then, resuspend the bead pellet in 25 .mu.L Buffer C1; then
transfer all into provided C2 tube (to activate the coupling group
on the beads). Mix gently on an orbital shaker for 15 min at room
temperature (25.degree. C.). Repeat with CML beads.
[0263] Wash the activated beads 2 times in 700 .mu.L Flow PBS,
magnetize the tube and remove supernatant after each wash.
Resuspend the activated beads in 50 .mu.L Flow PBS. Add 50 .mu.L of
the bait antibody (0.2-1.0 mg/mL stock concentration) to the
activated bead mix. Mix for 2 hours at 25.degree. C. by placing the
tube on a vibrating shaker or taped to a standard vortexer at low
shake setting. This will ensure sufficient mixing to prevent
settling of the beads. Overnight incubation is not necessary as
this kit is optimized for rapid coupling.
[0264] Wash Ab coupled beads 2 times in 700 .mu.L PBS, magnetize
the tube for Dynabeads, and remove supernatant after each wash.
Place the CML beads for 5 minutes of centrifugation at 5000 g.
Resuspend the coupled beads in 100 .mu.L Universal Buffer. These
can be stored at 4.degree. C. for at least one year. Resuspend the
beads well before use to ensure consistency for each
experiment.
[0265] Optically Active Agent Labeling on Antibodies and Optical
Reference Control Preparation.
[0266] Up to 150 .mu.g of antibody per reaction can be set up in
the provided glass vials. Antibody to dye ratio is 1:1. Total
reaction volume optimally should be at 110 .mu.L finally.
[0267] 1) Add 10 .mu.L Modifier into the 100 .mu.L of antibodies,
mix gently.
[0268] 2) Pipette all the mix into the lyophilized dye in the glass
vial, mix and incubate for 3 hours to overnight in dark at room
temperature, 25.degree. C.
[0269] 3) Add 1 .mu.L of quencher for every 10 .mu.L antibody used.
The conjugate can be used after 30 minutes of quenching.
For optical reference control preparation, follow the entire step
of "Coupling of bait antibodies to beads" with the conjugated
antibodies.
[0270] Performing Digital-Cell Western (DCW).
[0271] Count, and pellet 1.times.10.sup.6 cells for each sample to
perform DCW. 16% formaldehyde is added directly into the culture
medium, please note final formaldehyde concentration should be at
1.5% and incubated the cells for 10 min at 25.degree. C. or room
temperature (RT). Then, pellet the cells (use dissociation media
for adherent cells) by low speed centrifugation. Resuspend the cell
pellet by vortexing in 500 .mu.l ice-cold Methanol and incubated on
ice for 5 minutes. Cells can be stored at -80.degree. C. for long
term with minimum degradation.
[0272] To perform staining for target protein expression, cells
should be washed twice in 500 .mu.l cold Universal buffer then
resuspended with this buffer at 50 .mu.l. It is recommended to test
fidelity of antibodies, but standard guidelines is approximately 50
ng of optically active labeled antibodies should be added and
incubated for 30 min at 4.degree. C. Then wash the cells twice with
500 .mu.l cold Universal buffer. Finally, samples were resuspended
in 150 .mu.l FVA Buffer and analyzed by flow cytometer (as noted in
Performing FVA Scan)
[0273] Performing FVA Complex Capture.
[0274] While lysis method and optimal lysis conditions can vary in
some cases of transient protein-protein interactions being
investigated, the lysis buffer provided in this kit is designed to
meet most applications and is suitable in many cases. Each lysis
buffer is sufficient to perform 10 IPs, and contain most necessary
inhibitors. Alternatively, other lysis buffers in general are
compatible to be used with this kit.
[0275] Lysis of 30.times.10.sup.6 cells in 100 .mu.L fresh Lysis
buffer in a 1.5-mL microcentrifuge tube for 10-20 minutes on ice.
Scale the lysis volume as needed. Two sonication pulses into the
lysate are highly recommended for most applications. Cell debris
and nuclei can be removed by brief centrifuge of the lysate at 5000
g for 2 minutes at 4.degree. C. Keep the supernatant and discard
the pellet. Add 1.times.10.sup.5 of the coupled beads to the
lysate, (recommend using chimney-bottom 96 well plate for this
step). Use 50 .mu.L of the lysate to perform FVA for each sample.
The lowest volume can be at 5 .mu.L. Place on a vertical rotating
wheel 4 hours in a cold room, alternatively, overnight incubation
in cold room can be performed. Ensure proper mixing to prevent bead
settling.
[0276] Probing of Bead-Captured Protein with Optically
Active-Labeled Antibodies.
[0277] Wash the IP beads two times in 500 .mu.L ice-cold FVA
Buffer, magnetize and remove supernatant and transfer it into a
second vessel (tubes or plates). Centrifuge for 5 minutes at 5000
g, and remove supernatant, the pellet is the CML-analyte complex,
and perform two washes for Dynabeads and CML beads, magnetize and
centrifuge respectively and discard supernatant after each wash.
Resuspend the beads in 20 .mu.L FVA buffer. Add optically
active-labeled antibodies to the samples. Check with vendor's
recommendation on antibody concentration use. In most applications,
add 0.5 .mu.L of stock antibody solution (at 0.2-1.0 mg/mL) per
tube or well, and incubate for 35 minutes on ice on an orbital
shaker. Wash probed samples two times in 500 .mu.L ice-cold FVA
buffer, magnetize and remove supernatant after each wash. Resuspend
the beads in 200 .mu.L FVA buffer per sample. Samples are now ready
for FVA scan and analysis.
Performing FVA Scan
[0278] The supplied beads in this kit are about 5 .mu.m in
diameter, approximately half the diameter of a typical lymphocyte.
Set forward Scatter (FSC) amp gain to 480 and the side scatter
(SSC) voltage to 550 to register the population of bead events to
on scale. Both Dynabeads and CML beads should form distinct
clusters/population in a linear fashion, place the gate circle on
all distinct populations of beads. The settings and gate should be
adjusted to include only beads populations not cell debris which
are typically seen below the bead populations.
[0279] Use the default collection criterion, which for most
applications is 10,000-25,000 acquisition events. Adjustment of
this criterion to higher acquisition events may be needed for rare
target. The staining of the beads normally produces distinct mode
fluorescence intensity (MoFI) when visualized on a histogram mode,
change to Standard "log mode" for the use in fluorescence
channel(s) detection.
[0280] Prepare and use unlabeled, unconjugated beads as the first
sample to set the negative control photomultiplier tube (PMT)
voltage. Optionally, bright positive control for the second sample,
such as beads with optically active-conjugated antibodies (See
Preparation before starting). Initially, a run of internal optical
standards is necessary, for each flow cytometer used. This will
create a spectral compensation profile that can be used for
subsequent and future FVA scans. The flow cytometer is now ready to
acquire the FVA from the probed samples.
Performing Protein-Nucleic Acid Enrichment
[0281] Upon completion of flow sorting, population of beads can be
precipitated by applying magnetic field for Dynabeads and transfer
the supernatant to a new tube or plate and centrifuge at 5000 g for
5 minutes to pellet the CML beads then discard the supernatant. No
wash needed. Heat the beads to 90.degree. C. for 15 minutes, cool
on ice for 5 minutes then add 50 .mu.L of 1.times. Nucleic acids
recovery buffer mix to the captured beads and incubate at
55.degree. C. for 2 hour. Apply magnetic field on the Dynabeads
digested mix and centrifuge at 5000 g for the CML digested mix, and
transfer the supernatant into respective new tubes/plates. Add 5
.mu.L of 10.times. Proteinase inhibitor to the supernatant.
Optionally, standard DNA purification can be performed after this
step but the supernatant is suitable for direct use in applications
of MPS, allelic discrimination assays, quantitative PCR expression
analysis, and RNA/CHIP PCR assays.
Example C
Nuclear Assay--Localization
[0282] BRCA1 expression and localization were analyzed in isolated
primary B-Lymphoblastoid cells from individuals with BRCA1
mutations. The assays were performed as randomized, anonymized
samples. The cells were treated with Etoposide and UV radiation to
cause double-stranded DNA breaks. About 2 million cells were
formalin-fixed and methanol permealized. Each experiment included
three biological and three technical repeats. The cytoplasmic
fraction was removed by hypotonic salt lysis method followed by
nucleus enrichment by centrifugation. Flow staining hydration
buffer was used overnight at 4.degree. C. to increase the nucleus
ball diameter from 2-3 .mu.m to above 5-7 .mu.m, followed by
nuclear staining with optically-labeled primary antibodies. The
results demonstrated that after treatment with Etoposide and UV
radiation (FIG. 7(A)) or the X-Ray mimetic drug, Bleomycin (FIG.
7(B)), the localization of BRCA 1 to nuclear foci was markedly
lower among mutant samples compared to normal samples. The samples
were compared to IgG (i.e., negative) controls The bead internal
autofluorescence and selected size events were also compared. The
BRCA1 relative fluorescence intensity was normalized by dividing
with the specific events to the total gated events. An inverse
correlation (P<0.0001) was observed between BRCA1
localization/staining in the nuclear/pathogenicity upon DNA damage
drug treatment. All cell lines with mutations showed significantly
lower staining, whereas normal cell lines demonstrated strong
nuclear staining. The staining intensity denotes BRCA1 localization
into the nucleus for DNA double stranded repair (DSB). The
mutations in BRCA1 abrogated this localization and repair
mechanism.
[0283] The nuclear localization kit, and method herein, have been
developed for the preparation of enrichment of nuclear, whole-cell
and cytoplasmic extracts from cells or tissue. This kit provides
method that is simple, fast and effective to measure protein
activities within cytoplasmic and/or nuclear compartments of the
cell. The nuclear localization kit can be used to prepare intact
nuclear balls (cellular nuclei) to monitor tumor suppressor genes
activities in whole intact cell and intact cellular nucleus. The
nuclear compartment enrichment collected by this kit can be used
for a variety of standard protocols besides PrCo-IP, NACo-IP, FVA,
including electrophoretic mobility shift assay (EMSA), DNA
footprinting, Western blotting and preparative purification of
nuclear proteins.
[0284] Each kit provides reagents for direct lysis of cells. First,
the cells are collected in ice-cold PBS in the presence of
phosphatase inhibitors or fixation (e.g. by 2% paraformaldehyde) to
limit further protein modifications (expression, proteolysis,
dephosphorylation, etc.). Then, the cells are resuspended in
hypotonic lysis buffer and strong detergent added which causes
breakage of the cells resulting in leakage of cytoplasmic proteins
into the supernatant. After collection of the cytoplasmic fraction,
the nuclei are fixed-permeabilized with, e.g., a methanol-based
storage buffer.
[0285] To prepare whole-cell extracts, cells are collected in the
PBS/phosphatase Inhibitors solution and lysed in the lysis buffer.
Solubilized proteins are separated from the cell debris by
centrifugation. The protein concentration of the cell extract can
be normalized by counting number of live cells during cell
collection, no protein quantification necessary. Optionally, a
Bradford assay can be used at this step. The method or kit can be
used to obtain nuclear, cytoplasmic or whole-cell extract from
cells or from tissue.
[0286] Successful extraction has been performed with NT2/D1, PC3,
LNCaP, DU145, and B-lymphoblastoid cells. In an embodiment, the kit
used comprises the components as follows:
TABLE-US-00001 Lysis Buffer AM1 (e.g. 10 ml) 1M Dithiothreitol
(DTT) (e.g. 500 .mu.l) Protease Inhibitor mix (e.g. 500 .mu.l) 10X
PBS (e.g. 4 .times. 100 ml) Phosphatase Inhibitors (e.g. 4 .times.
50 ml) 10X Hypotonic Buffer (e.g. 50 ml) Detergent (NP40 mix) (e.g.
5 ml).
Protocol Examples:
[0287] The following exemplary protocol is based on samples of
approximately 20.times.10.sup.6 cells, which corresponds to 10
wells on a 96-well plate. Each well is one reaction. Prepare
PBS/phosphatase inhibitors, hypotonic buffer and total lysis
buffer. Place buffers and any tubes needed on ice before beginning
assay.
[0288] Step 1: Cell Collection
1. Aspirate media out of culture vessel. Wash with 5 ml ice-cold
PBS/Phosphatase Inhibitors. Aspirate solution out and add 3 ml
ice-cold PBS/Phosphatase Inhibitors. 2. Remove cells from
flask/dish by gently scraping with cell lifter for adherent cells.
Transfer cells to a sterile 50 ml conical tube. 3. Centrifuge cell
suspension for 5 minutes at 500 rpm in a centrifuge pre-cooled at
4.degree. C. 4. Discard supernatant. Keep cell pellet on ice.
[0289] Step 2: Cytoplasmic Fraction Collection
1. Gently resuspend cells in 5 ml 1.times. Hypotonic Buffer with
250 .mu.l NP40 detergent by pipetting up and down several times.
Transfer to a pre-chilled microcentrifuge tube. Incubate for 15
minutes on ice. 2. Vortex 10 seconds at highest setting. 3.
Centrifuge suspension for 30 seconds at 2-3,000.times.g in a
centrifuge pre-cooled at 4.degree. C. 4. Transfer supernatant
(cytoplasmic fraction) into a pre-chilled microcentrifuge tube. (If
you began working from tissue, combine this supernatant with that
obtained in Step 1, No. 3 of the Nuclear enrichment protocol for
tissue.) Store the supernatant at -80.degree. C. until ready to
use. Use the pellet for nuclear collection.
[0290] Step 3: Nuclear Enrichment Collection and Nuclear
Extracts.
1. Resuspend nuclear pellet in fixation and permeabilizing buffer
or 500 .mu.l Total lysis buffer by pipetting up and down. Vortex 10
seconds at highest setting. 2. Incubate suspension for 30 minutes
on ice on a rocking platform set at 150 rpm. 3. Vortex 30 seconds
at highest setting. Centrifuge for 10 minutes at 14,000.times. g in
a microcentrifuge pre-cooled at 4.degree. C. Transfer supernatant
(nuclear fraction) into a pre-chilled microcentrifuge tube. 4.
Aliquot and store at -80.degree. C. Avoid freeze/thaw cycles.
[0291] Starting from Tissue:
Step 1: Tissue Homogenization
[0292] 1. Weigh tissue and dice into very small pieces using a
clean razor blade. Collect pieces in a pre-chilled, clean MP
homogenizer Fastprep 24 sample prep tubes (6900 series). 2. On ice,
add 3 ml ice-cold 1.times. hypotonic buffer supplemented with DTT
and Detergent (3 .mu.l of the provided 1 M DTT and 3 .mu.l of the
provided detergent) per gram of tissue and homogenize. Incubate on
ice for 15 minutes. 3. Centrifuge for 10 minutes at 850.times. g at
4.degree. C. Transfer the supernatant into a pre-chilled
microcentrifuge tube. (Save this supernatant and pool it with the
supernatant that will be collected later in Step 2, No. 3 of the
Nuclear enrichment protocol for cells.) 4. At this point, the
tissue is homogenized. However, most of the cells are not yet
lysed. Therefore, continue the procedure with the cell pellet at
Step 2, No. 1 of the Nuclear enrichment protocol for cells, based
on a 20.times.10.sup.6 cells.
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