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SIGMA-ALDRICH
FLAG® 96-well Immunoprecipitation System
User Manual
®
SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
FLAG and ANTI-FLAG are registered trademarks of Sigma-Aldrich Biotechnology LP. The product designations of pFLAG,
p3XFLAG, pFLAG-1, pFLAG-2, pFLAGSHIFT, pFLAG-CTS, pFLAG-ATS, pFLAG-MAC, pFLAG-CMV, YEpFLAG, and FLAG-
BAP are trademarks of Sigma-Aldrich Biotechnology LP.
All other trademarks are the property of their respective owners.
© 2010, Sigma-Aldrich Biotechnology LP. All rights reserved.
Date 2 Version #
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
1.0 TABLE OF CONTENTS
1.0 Table of Contents................................................................................................................................3
2.0 System Components ..........................................................................................................................4
Immunoprecipitation Vector Kit (Product # COIP-P) ............................................................................4
FLAG 96-well Immunoprecipitation Kit (Product # HT-COIP-1) ...........................................................4
Immunoprecipitation Detection Kit (Product # COIP-D) .......................................................................4
3.0 Introduction .........................................................................................................................................5
4.0 FLAG 96-well Immunoprecipitation System Overview ...................................................................6
5.0 Immunoprecipitation Vector Kit (Product # COIP-P).......................................................................7
5.1 Reagents Provided (sufficient for 96 samples)............................................................................7
5.2 Additional Reagents and Equipment (Not Supplied) ...................................................................7
5.3 Expression Vectors......................................................................................................................8
5.4 Control Vectors ..........................................................................................................................10
5.5 Verification Primers ...................................................................................................................11
6.0 FLAG 96-well Immunoprecipitation Kit (Product # HT-COIP-1) ...................................................12
6.1 Reagents Provided (sufficient for 96 samples)..........................................................................12
6.2 Additional Reagents and Equipment (Not Supplied) .................................................................12
7.0 Immunoprecipitation Detection Kit (Product # COIP-D) ...............................................................13
7.1 Reagents Provided (sufficient for 96 samples)..........................................................................13
7.2 Additional Reagents and Equipment (Not Supplied) .................................................................13
8.0 Protocols for Studying Protein-Protein Interactions with the ANTI-FLAG M2 Plate..................14
8.1 Overview of the Analysis of Protein-Protein Interaction with the ANTI-FLAG M2 Plate by
ELISA or Western blotting .........................................................................................................14
8.2 General Notes ...........................................................................................................................15
8.2.1Plasmid DNA for transfection .............................................................................................15
8.2.2Transfection reagent ..........................................................................................................15
8.2.3Cell lysates used for co-immunoprecipitation assay..........................................................15
8.2.4Detection of endogenous binding partners during Western blotting or ELISA ..................16
8.3 Preparation of Cell Extract.........................................................................................................16
8.4 Immunoprecipitation with ANTI-FLAG M2 plate ........................................................................16
8.5 Detection of the Bound Protein by ELISA or Western Blotting..................................................17
8.5.1ELISA .................................................................................................................................17
8.5.2Western Blotting .................................................................................................................18
9.0 Troubleshooting Guide ....................................................................................................................19
10.0 References.........................................................................................................................................20
Appendix 1: Construction of fusion genes using standard cloning techniques................................21
Appendix 2: Screening of recombinant plasmids by colony PCR .......................................................22
Procedure for Screening Recombinant Plasmids...............................................................................22
Appendix 3: Capturing the Amino-terminal FLAG-BAP Fusion Protein with the ANTI-FLAG M2
plate and evaluating the pNPP substrate integrity ................................................................................24
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
2.0 SYSTEM COMPONENTS
Immunoprecipitation Vector Kit (Product # COIP-P)
• pFLAG-CMV™-2 Expression Vector (10 µg)
• pc-Myc-CMV-2 Expression Vector (10 µg)
• pFLAG-CMV-2-p53 control plasmid (20 µg)
• pc-Myc-CMV-2-Large T antigen control plasmid (10 µg)
• pc-Myc-CMV-2-BAP control plasmid (10 µg)
• Verification Primer-MF (2 nmol)
• Verification Primer-MR (2 nmol)
• Technical Bulletins
Storage Conditions: Store all components at -20°C
FLAG 96-well Immunoprecipitation Kit (Product # HT-COIP-1)
• ANTI-FLAG M2 High Sensitivity Capture Plate (1 plate)
• Amino-terminal FLAG-BAP Fusion Protein (2.5 µg)
• 10× Wash Buffer (50 ml)
• Lysis Buffer (50 ml)
• 2× Sample Buffer (6 ml)
• SealPlate Film (6)
• User Manual
Storage Conditions: Store all components at 4°C
Immunoprecipitation Detection Kit (Product # COIP-D)
• Monoclonal Anti-c-Myc, Clone 9E10, Alkaline Phosphatase Conjugate (0.25 ml)
• Tris-Buffered Saline, pH 8.0, with 3% Nonfat Milk (1 packet)
• SIGMA FAST™ p-Nitrophenyl Phosphate Tablets (5 tablet sets)
• Technical Bulletins
Storage Conditions: Store all components at 4°C
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
3.0 INTRODUCTION
Researchers studying protein-protein interactions commonly use one of several different two-hybrid
systems. While these systems are of some use in determining protein binding partners, all of the
current two-hybrid systems generate false positives. Sigma's FLAG-96-well Immunoprecipitation Kit
provides a unique method to validate protein-protein interactions in a convenient 96-well format. This
system enables potential binding partners for the protein of interest to be tagged with FLAG and c-
Myc epitopes, followed by capture and detection using the ANTI-FLAG M2 High Sensitivity Capture
Plate. The system offers the following advantages compared to currently available techniques:
1. Rapid 96-well format—The 96-well plate configuration is very effective at handling multiple
samples and greatly increases the throughput of the assay. Additionally, this is an ideal
system for optimizing in vitro binding conditions.
2. No leaching of ANTI-FLAG M2 antibody—The ANTI-FLAG M2 antibody is covalently
coupled to the surface of the well and the elution conditions used are relatively mild, so very
little antibody leaches from the plate. This results in little to no interference from denatured
antibody heavy and light chain bands during Western blot analysis.
3. Rapid protocol with quantitative results—Because an ELISA format can be used with this
system, the protein-protein interaction assay is rapid (less than 4 hours), sensitive, and
quantitative.
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
4.0 FLAG 96-WELL IMMUNOPRECIPITATION SYSTEM OVERVIEW
Figure 1. Outline of the procedure
Clone two potentially interacting proteins into the two mammalian epitope-tagged expression vectors provided in the
Immunoprecipitation Vector Kit. One vector generates an N-terminally tagged FLAG fusion protein while the other vector
produces an N-terminally tagged c-Myc fusion protein.

Co-express the proteins in appropriate mammalian host cells

Prepare cell extracts with Lysis Buffer

Dilute the cell lysate with 1× Wash Buffer (if necessary)

Incubate cell lysate in the 96-well ANTI-FLAG M2 High Sensitivity Capture Plate

Wash the ANTI-FLAG M2 High Sensitivity Capture Plate with 1× Wash Buffer

Analyze captured proteins by ELISA or Western blotting
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
5.0 IMMUNOPRECIPITATION VECTOR KIT (PRODUCT # COIP-P)
This kit includes the transient mammalian expression vectors pFLAG-CMV™-2 and pc-Myc-CMV-2,
which are designed for the construction and expression of N-terminally tagged fusion proteins with a
1 2
FLAG epitope (DYKDDDDK) or a c-Myc epitope (EQKLISEEDL) , respectively. The control plasmids
pFLAG-CMV-2-p53, pc-Myc-CMV-2-Large T antigen and pc-Myc-CMV-2-BAP are also supplied. The
control plasmids can be used as positive- and negative-binding partners in the immunoprecipitation
analysis (see Section 5.4). Figures 2 through 4 and Table I illustrate the features of the supplied
vectors.
5.1 Reagents Provided (sufficient for 96 samples)
Part Product # Amount
pFLAG-CMV™-2 Expression Vector E7398 1 × 10 μg
pc-Myc-CMV-2 Expression Vector P9236 1 × 10 μg
pFLAG-CMV™-2-p53 Control Plasmid P9986 1 × 20 μg
pc-Myc-CMV-2-Large T antigen Control Plasmid P9861 1 × 10 μg
pc-Myc-CMV-2-BAP Control Plasmid P9736 1 × 2.5 μg
Verification Primer-MF P2987 1 × 2 nmol
Verification Primer-MR P3112 1 × 2 nmol
5.2 Additional Reagents and Equipment (Not Supplied)
• Pipets (single- or multi-channel) • Thermal cycler
• Pipette tips • PCR tubes or multi-well PCR plate
• Microcentrifuge tubes • Taq DNA polymerase
• Microcentrifuge • Restriction enzymes
• Reagent reservoir • Phosphatase suitable for DNA
• Vortexer • Competent E. coli cells
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• MilliQ or sterile water • LB-ampicillin agar plates
• DNA plasmid purification kit • LB medium with 100 μg/ml Ampicillin
• Transfection reagent • 15 ml culture tubes
• Agarose gel electrophoresis system • 37°C incubator
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
5.3 Expression Vectors
Figure 2. Circular maps of the expression vectors
4679 Hind III 4688 Hind III
Not I Not I
4227 166 4236 166 EcoR I
EcoR I
Cla I Cla I
f1 IG Bgl II f1 IG Bgl II
EcoR V EcoR V
CMV Kpn I CMV Kpn I
Sal I Sal I
Xba I Xba I
FLAG Bam HI c-Myc Bam HI
Sma I Sma I
pFLAG-CMV™-2 pc-Myc-CMV-2
4.7 kb 4.7 kb
hGH poly A hGH poly A
1642 1651
SV40 ori SV40 ori
E.coli ori/b-lact 2908 E.coli ori/b-lact 2917
Table I. Expression vector features
pFLAG-CMV™-2 pc-Myc-CMV-2
Feature
(4679 bp) (4688 bp)
CMV promoter 166-916 166-916
Verification Primer-MF 825-854 825-854
Translational initiation 932-934 932-934
FLAG and c-Myc sequences 935-958 935-967
Multiple cloning region 956-1022 965-1031
hGH poly A 1023-1642 1032-1651
Verification Primer-MR 1080-1103 1089-1112
SV40 ori 1661-2005 1670-2014
pBR322 ori/beta-lactamase 2908-4092 2917-4101
f1 intragenic region 4227-4679 4236-4688
Note: The only difference between the two expression vectors is the epitope tags.
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
Figure 3. Nucleotide sequences of the epitope tags and multiple cloning region of
the pFLAG-CMV™-2 (upper) and pc-Myc-CMV-2 (lower) expression vectors
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
5.4 Control Vectors
The Immunoprecipitation Vector Kit provides positive and negative control vectors for co-
immunoprecipitation (Figure 4). pFLAG-CMV™-2-p53 Control Plasmid expresses the N-terminally-
tagged FLAG fusion protein containing the wild-type C-terminal domain of mouse p53 (residues 73-
3
390). pc-Myc-CMV-2-Large T antigen Control Plasmid expresses the N-terminally-tagged c-Myc
4
fusion protein containing the C-terminal domain of Large T-antigen (residues 86-708). pc-Myc-CMV-
2-BAP Control Plasmid expresses the N-terminally-tagged c-Myc fusion protein containing bacterial
alkaline phosphatase (BAP). Mammalian cells co-transfected with control plasmids pFLAG-CMV™-2-
p53 and pc-Myc-CMV-2-Large T antigen will express tagged fusion proteins that interact, providing a
5
positive control for the co-immunoprecipitation analysis. Cells co-transfected with pFLAG-CMV™-2-
p53 and pc-Myc-CMV-2-BAP express tagged fusion proteins that do not interact, demonstrating a
negative result in the co-immunoprecipitation analysis.
Figure 4. Circular maps of control vectors
5649
5197 166 Hind III
f1 IG p53
CMV
coding
sequence
FLAG
pFLAG-CMV™-2-p53 Xba I
Control Plasmid
hGH poly A
5.6 kb
2612
SV40 ori
E.coli ori/b-lact 2631
6838 6133
6386 166 Xba I 5681 166 Hind III
f1 IG Large T f1 IG BAP
CMV
antigen CMV
coding
coding sequence
c-Myc
sequence c-Myc
pc-Myc-CMV-2-Large T antigen Xba I pc-Myc-CMV-2-BAP Sma I
Control Plasmid Control Plasmid
6.8 kb hGH poly A 6.1 kb hGH poly A
3801 3096
SV40 ori SV40 ori
E.coli ori/b-lact 5067 E.coli ori/b-lact 4362
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
5.5 Verification Primers
Verification primer-MF and Verification primer-MR are supplied with this kit and allow the researcher
to screen for the presence, orientation, and reading frame of the DNA insert by colony PCR or DNA
sequencing (Table II).
Table II. Vector primers and sequences
Vector primers Product #. Sequence
Verification Primer-MF P2987 5’ AAT GTC GTA ATA ACC CCG CCC CGT TGA CGC 3′
Verification Primer-MR P3112 5’ TAT TAG GAC AAG GCT GGT GGG CAC 3′
Notes:
1. Please see Appendix 1 for procedures involved in cloning recombinant plasmids.
2. Please see Appendix 2 for procedures involved in screening recombinant plasmids by
Colony PCR.
3. If the genes of interest are already cloned into expression vectors that contain FLAG or c-Myc
tags, it is not necessary to subclone them into the expression vectors provided in this kit.
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
6.0 FLAG 96-WELL IMMUNOPRECIPITATION KIT (PRODUCT # HT-COIP-1)
The FLAG-96-well Immunoprecipitation Kit provides all of the unique reagents required to study
protein-protein interactions using a 96-well ANTI-FLAG M2 High Sensitivity Capture Plate (ANTI-
FLAG M2 plate). The ANTI-FLAG M2 plate is designed to capture recombinant FLAG fusion proteins
obtained from cell lysates of transfected cells.
6.1 Reagents Provided (sufficient for 96 samples)
Part Composition Product # Amount
ANTI-FLAG M2 High Sensitivity
— P2983 1 plate
Capture Plate
20 mM Tris-HCl (pH 7.4), 1 M NaCl, 1 mM DTT,
Lysis Buffer L1413 50 ml
1.0% Triton® X-100
10× Wash Buffer 500 mM Tris-HCl (pH 7.4), 1.5 M NaCl W4511 50 ml
125 mM Tris-HCl (pH 6.8), 4% SDS, 20% (v/v)
2× Sample Buffer B2556 6 ml
glycerol, 0.004% bromphenol blue
Amino-terminal FLAG-BAP Fusion
— A1974 2.5 μg
Protein
SealPlate Film — Z36, 965-9 5
6.2 Additional Reagents and Equipment (Not Supplied)
• Pipets (single- or multi-channel) • Tissue culture incubator
• Pipette tips • Mammalian protease inhibitor cocktail
• Microcentrifuge tubes • Ice-cold PBS
• Reagent reservoir • Cell scrapers
• Microcentrifuge at 4°C • Kit for measuring total protein
• Vortexer concentration
• MilliQ or sterile water • 4°C incubator
• Tissue culture plates • Plate washer
• Tissue culture medium • Rotary shaker
• Tissue culture cells, such as COS-7
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
7.0 IMMUNOPRECIPITATION DETECTION KIT (PRODUCT # COIP-D)
The Immunoprecipitation Detection Kit provides all of the unique reagents required to detect protein-
protein interactions using a plate-based colorimetric assay. The substrate p-Nitrophenyl Phosphate
Disodium (pNPP) is hydrolyzed by Anti-c-Myc Alkaline Phosphatase-conjugated antibody to produce
a yellow-colored solution that can be read spectrophotometrically at 405 nm. Tris Buffered Saline (pH
8.0) with 3% Nonfat Milk is used to dilute the Anti-c-Myc Alkaline Phosphatase Conjugate. The pNPP
reaction is terminated with 3 N NaOH and then read at 405 nm. The SIGMA FAST™ pNPP tablet set
provided in the kit produces a ready-to-use solution after the addition of 5 ml of distilled or deionized
water.
7.1 Reagents Provided (sufficient for 96 samples)
Kit Components Product # Quantity
Monoclonal Anti-c-Myc, Clone 9E10, Alkaline Phosphatase Conjugate A5963 1 × 0.25 ml
Tris Buffered Saline, pH 8.0, with 3% Nonfat Milk T8793 1 packet
SIGMA FAST™ p-Nitrophenyl Phosphate Tablets N1891 5 tablet sets
7.2 Additional Reagents and Equipment (Not Supplied)
• Pipets (single- or multi-channel) • β-Mercaptoethanol
• Pipette tips • Bulldog clamps
• Microcentrifuge tubes • Boiling water bath
• Reagent reservoir • SDS-PAGE gels and apparatus
• Microcentrifuge at 4°C • Protein transfer apparatus
• Vortexer • Protein transfer membrane
• Plate washer • Tris-glycine buffer
• Plate mixer • TBST
• Rotary shaker • TBS
• MilliQ or sterile water • Anti-mouse IgG peroxidase conjugate
• 3 N NaOH • ANTI-FLAG M2 monoclonal antibody
• 50 ml conical tubes peroxidase (HRP) conjugate
• Spectrophotometer capable of • ECL Plus™ Reagent
measuring absorbance at 405 nm
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
8.0 PROTOCOLS FOR STUDYING PROTEIN-PROTEIN
INTERACTIONS WITH THE ANTI-FLAG M2 PLATE
8.1 Overview of the Analysis of Protein-Protein Interaction with the ANTI-
FLAG M2 Plate by ELISA or Western blotting
Day 1: Plate cells

Day 2: Transfect cells

Day 3 or 4: Harvest cells with Lysis Buffer supplemented with mammalian protease inhibitor cocktail and prepare cell lysate

Incubate cell lysate with the ANTI-FLAG M2 plate for 1 hour at 4°C
↓ (diluting the cell lysate is optional)
Wash the plate 4 times with 200 μl (per well) of the 1× Wash Buffer
After this step, the plate can be analyzed by

ELISA or Western blotting
Analysis with ELISA: Analysis with Western Blotting:
Add 200 μl of Anti-c-Myc Alkaline Phosphatase Conjugate Add 60 μl of 2× Sample Buffer (supplemented with
(diluted 1:100 in Blocking Buffer) to each well and incubate 10% β-Mercaptoethanol) to each well and incubate at room
for 1 hour at room temperature on a rotary shaker temperature for 30 minutes (with shaking on a vortexer)

Wash the plate 4 times with 200 μl (per well) 1× Wash Buffer ↓

Transfer the contents of each well to microcentrifuge tube
Incubate each well with 200 μl pNPP substrate
and boil for 5 minutes

Incubate at room temperature for 15 to 30 min or until the

yellow color develops

Stop the reaction with 3 N NaOH

Western Blotting
Measure A405 with a plate reader
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8.2 General Notes
Please read this section carefully before proceeding with the experiment.
8.2.1 Plasmid DNA for transfection
To obtain maximum transfection efficiency, the DNA used for transfections must be of high quality
(free of protein, RNA, and chemical contamination, with an A260:A280 ratio of 1.8-1.9). To produce
high-quality plasmid DNA, we recommend Sigma’s GeneElute Endo-Free Plasmid Purification Kit
(Product # PLEX 15).
8.2.2 Transfection reagent
The quality of a transfection reagent dictates the sensitivity and specificity of an assay using the
FLAG 96-well Immunoprecipitation Kit. Generally, high transfection efficiency yields a more sensitive
assay, requiring a smaller sample volume. The Immunoprecipitation Vector Kit includes three control
plasmids for optimizing and monitoring the efficiency of transfection. To obtain high-efficiency
transfections, we recommend Sigma’s ESCORTII Transfection Reagent (Product # L6037) for
commonly used cell lines.
8.2.3 Cell lysates used for co-immunoprecipitation assay
1. Expression level of the fusion proteins in the cell lysate. The concentration of the
expressed fusion protein is critical to the success of the co-immunoprecipitation analysis. The
expression of the FLAG and c-Myc fusion proteins should be verified by Western blotting with
ANTI-FLAG M2 antibody and anti-c-Myc antibody, prior to the co-immunoprecipitation
analysis. In general, a high concentration of fusion protein in the cell lysate will yield more
sensitive results. For each tag, the control plasmids supplied in the Immunoprecipitation
Vector Kit can be used as positive controls in transfection and Western blot analysis. FLAG-
p53 can be detected with ANTI-FLAG M2 antibody, while the c-Myc-Large T antigen and c-
Myc-BAP can be detected with monoclonal anti-c-Myc antibody 9E10.
2. Amount of cell lysate used in the co-immunoprecipitation assay. The optimum amount
of cell lysate added to the wells of the ANTI-FLAG M2 plate may vary with each experimental
system and should be determined empirically. The ANTI-FLAG M2 plate-based immuno-
precipitation is very sensitive, especially in ELISA. When analyzing the interaction of FLAG-
p53 and c-Myc-Large T antigen, the c-Myc-Large T antigen can be detected in as little as 5
μg of the cell lysate from co-transfected COS-7 cells (as compared to the negative control cell
lysate).
Note: The volume of the cell lysate used should not exceed 200 μl since the wells of
the ANTI-FLAG M2 plate are coated up to the 200 μl level with the ANTI-FLAG
M2 antibody.
3. Dilution of the cell lysate. The relative binding affinity for different protein complexes may
vary. Since high salt and detergent levels are present in the Lysis Buffer, it may be necessary
to dilute the cell lysate with 1× Wash Buffer to minimize disruption of the protein-protein
interactions.
Note: For the positive control interaction between FLAG-p53 and c-Myc-Large T
antigen, dilution of the cell lysate is not necessary.
4. Negative-binding protein partner. Inclusion of a negative-binding partner along with the
FLAG-tagged protein construct is critical for verifying the specific protein-protein interaction
observed. Over-expression of a fusion protein may cause nonspecific protein-protein
interactions between the proteins being tested. The c-Myc-BAP vector provided in the
Immunoprecipitation Vector Kit can be used as a negative-binding protein partner.
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SIGMA-ALDRICH FLAG 96-well Immunoprecipitation System User Manual
8.2.4 Detection of endogenous binding partners during Western blotting or ELISA
Although this kit and protocol are designed for detecting the c-Myc-tagged binding partner from co-
transfected cell lysates using anti-c-Myc antibody, you may use the ANTI-FLAG M2 plate to capture
endogenous binding partners that interact with the FLAG-fusion protein from cells transfected only
with the FLAG-fusion protein. In this situation, optimum dilution and incubation times should be
determined for the specific antibodies used to detect the endogenous binding partners during
Western blotting or ELISA. Since the ANTI-FLAG M2 plate is coated with monoclonal ANTI-FLAG M2
antibody, either polyclonal antibody or antibody conjugates should be used for ELISA-based detection
to avoid problems with cross-reactivity.
Note: This protocol has been optimized for analyzing the interaction of FLAG-p53 and c-Myc-
Large T antigen expressed in COS-7 cells. To ensure that the system works in your
hands, we recommend including the control plasmids provided in the
Immunoprecipitation Vector Kit in your experiment.
8.3 Preparation of Cell Extract
1. Aspirate growth medium from the


Use: 0.5362