Enabling Epigenetics Research

Universal Magnetic Co-IP Kit
Catalog No. 54002(Version A4)

Copyright 2014 Active Motif, Inc.

Revision

Date

Description of Change

A4

March, 2024

Important: Phosphatase Inhibitors (AM Part 102146 ) has been
replaced with 100x Phosphatase Inhibitors (AM Part 105840).
Consult the appropriate protocol (nuclear, cytoplasmic or whole-cell
extract from cells or from tissue) to determine the updated amountof PBS/Phosphatase Inhibitors solution that will be required foryour extraction(s).

Information in this manual is subject to change without notice and does not constitute a
commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any
warranty of any kind, either explicit or implied. Information may be changed or updated inthis manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated,
in whole or in part, without the prior written consent of Active Motif, Inc. This documentation
is proprietary information and protected by the copyright laws of the United States andinternational treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2014 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 100; Carlsbad, CA 92008. All
rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to theirrespective companies.

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Contents
Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
Kit Components and Storage  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Protocols – Nuclear Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Quick Chart for Preparing Buffers for Nuclear Extraction from Cells . . . . . . . . . . .  5
Nuclear Extraction from Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
Nuclear Extraction from Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Protocols – Whole-cell Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Quick Chart for Preparing Whole-cell Extraction Buffers . . . . . . . . . . . . . . . . . . . . .  8
Whole-cell Extraction from Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  9
Whole-cell Extraction from Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Protocols – Co-Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Co-Immunoprecipitation Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Western Blotting Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section A. Use of Magnetic Beads and Included Bar Magnet . . . . . . . . . . . . . . . . 15
Section B. Troubleshooting Guide  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

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Overview
Co-Immunoprecipitation (Co-IP) is a powerful method used to study protein/protein
interactions. In Co-IP, one antibody is used to immunoprecipitate a target antigen, whichat the same time co-precipitates any other proteins that are bound to/interacting with thefirst protein. These protein complexes are then captured by antibody binding beads (suchas Protein A or G). This is followed by elution of the antibody-bound proteins, SDS-PAGE
and Western blot using a second antibody that is targeted against one of the bound,interacting proteins.
However, traditional methods for performing Co-IP are not optimal for studying DNA-binding
protein complexes as the complexes are often disrupted during the extraction process.
And, high background has often necessitated the use of high-stringency buffers that,while reducing background, can destroy the protein complexes before they have been
immunoprecipitated. In addition, traditional high-salt extraction procedures do not remove
histone proteins from DNA.
Because of this, Active Motif’s Universal Magnetic Co-IP Kit has been developed to enable
the preparation and immunoprecipitation of protein complexes from both nuclear andwhole-cell extracts, beginning from either cell or tissue samples. The kit utilizes protein
G-coated magnetic beads that improve Co-IP by simplifying the IP and wash steps whileproviding a much lower background. This reduced background enables the use of a lowstringency Co-IP/Wash Buffer that helps maintain the protein complexes. To further improve
Co-IP of nuclear complexes, the kit’s proprietary Enzymatic Shearing Cocktail uses DNA
digestion to release undissociated protein complexes from the DNA. Even histone complexes
are released, and they remain intact. Moreover, all extraction and IP steps, both nuclear
and whole-cell, are performed in buffers supplemented with Protease, Phosphatase andDeacetylase Inhibitors, which limits further protein modifications (expression, proteolysis,
dephosphorylation, etc.), thereby preserving the in vivo states of the immunoprecipitatedprotein complexes.
Each Universal Magnetic Co-IP Kit provides sufficient reagents to perform 25 nuclear and 25
whole-cell extractions, and then to perform 25 co-immunoprecipitations. The kit includes
optimized nuclear and whole-cell protein complex extraction buffers, inhibitor solutions,immunoprecipitation/wash buffer, Magnetic Protein G Beads, a powerful Bar Magnet for
creating a magnetic stand and a comprehensive protocol to provide a simplified method to
capture and detect protein complexes.
Product

FormatCatalog No.

Universal Magnetic Co-IP Kit

25 rxns

54002

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1

Kit Performance and Benefits

Figure 1: Nuclear Co-IP of SRC-1 and ERα.
The Universal Magnetic Co-IP Kit was used to make nuclear extract from MCF-7 cells induced 1 hourwith 10 nM Estradiol. IP was performed on 300 µg samples using 2 µg of SRC-1 pAb, ERα pAb andrabbit IgG (as a negative control). Western blot was then performed using the ERα pAb on 10 µg
Input Extract, SRC-1 IP, ERα IP and the rabbit IgG IP.

Figure 2: Detection of acetylated Histone H3.
HeLa nuclear extracts were made using the Universal Magnetic Co-IP Kit and a traditional high-salt
extraction protocol, supplemented with 1 µM trichostatin A, a deacetylase inhibitor. Five and ten µg
samples of each extract were used in Western with Histone H3 acetyl rabbit pAb (Cat No. 39139).
Protein was detected only in samples made using the kit’s nuclear extraction procedure, as it was
designed to release histone and other protein complexes from DNA while preserving modifications.

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Kit Components and Storage
Kit components can be stored at -20ºC prior to first use. Then, we recommend storing eachcomponent at the temperatures recommended in the table below:
Kit Component

Quantity

Storage

10X PBS

80 mL

4°C for 6 months

10X Hypotonic Buffer

50 mL

4°C for 6 months

Whole-cell Lysis Buffer

10 mL-20°C for 6 months

Co-IP/Wash Buffer

100 mL4°C for 6 months

Detergent

3 mL

4°C for 1 year

Phosphatase Inhibitors

9 mL-20°C for 6 months

500 µL-20°C for 6 months

5 mL-20°C for 6 months

100 mM PMSF

500 µL-20°C for 6 months

0.5 M EDTA

200 µL4°C for 6 months

Digestion Buffer

10 mL

4°C for 6 months

Enzymatic Shearing Cocktail

50 µL-20°C for 6 months

Protein G Magnetic Beads*

650 µL4°C for 6 months

Protease Inhibitor Cocktail
Deacetylase Inhibitor

Bar Magnet
Mini Glue Dots

1RT

1 sheetRT* The Protein G Magnetic Beads are shipped on dry ice, but should not be re-frozen by thecustomer. Upon receipt, the beads should be stored at 4ºC.

Additional Materials Required• 5 and 10 mL pipettes and pipettors• Cell scraper• 15 mL conical tubes• Microcentrifuge tubes• Centrifuge (with swinging buckets adapted to 15 mL conical tubes) and microcentrifuge
pre-cooled to 4°C• Rocking platform• Distilled water• Apparatus to rotate tubes end-to-end at 4°C (for co-immunoprecipitation)• Antibodies for IP and Western blot
• 37°C water bath
• Reagents and equipment for performing SDS-PAGE and Western blot

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3

Protocols – Nuclear Extraction
Buffer Preparation and Recommendations
Imporant: As of May, 2022, there is a new formulation of Phosphatase Inhibitors. The
originial Phosphatase Inhibitors (AM Part 102146), have been replaced by 100x Phosphatase
Inhibiors (AM Part 105840). Consult the appropriate protocol (nuclear, cytoplasmic or wholecell extract from cells or from tissue) to determine the updated amount of PBS/Phosphatase
Inhibitors solution that will be required for your extraction(s).
Note:Phosphatase Inhibitors, PMSF and PIC lose activity 24 hours after dilution.
Therefore, use all of the solutions below on the same day prepared, thendiscard any remaining solution.

Preparation of PBS/Inhibitors (8 mL/plate)
To make nuclear extract from a 100 mm plate of cells, prepare 8 mL of PBS/Inhibitors solution
as follows: mix 0.8 mL 10X PBS in 7.04 mL distilled water, then add 80 µL Phosphatase
Inhibitors and 80 µL Deacetylase Inhibitor. Vortex to mix and place on ice. (This buffer is used
for both nuclear and whole-cell extractions.)
Preparation of Complete Hypotonic Buffer (500 µL/plate)
To make nuclear extract from a 100 mm plate of cells, prepare 500 µL Complete Hypotonic
Buffer as follows: mix 50 µL 10X Hypotonic Buffer, 435 µL distilled water, 5 µL Phosphatase
Inhibitors, 5 µL Deacetylase Inhibitor, 2.5 µL Protease Inhibitor Cocktail (PIC) and 2.5 µL 100
mM PMSF. Vortex to mix and place on ice.
Preparation of Complete Digestion Buffer (100 µL/plate)
To make nuclear extract from a 100 mm plate of cells, prepare 100 µL of Complete
Digestion Buffer as follows: mix 97 µL Digestion Buffer with 1 µL Phosphatase Inhibitors, 1µL Deacetylase Inhibitor, 0.5 µL Protease Inhibitor Cocktail (PIC) and 0.5 µL 100 mM PMSF.
Vortex to mix and place on ice.

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4Quick Chart for Preparing Buffers for Nuclear Extraction from Cells
60mm plate or 100mm plate or
3.2 x 106 cells
8.8 x 106 cells

150mm plateor 2 x 107 cells

Reagents to prepare

Components

PBS/Phosphatase
Inhibitors

10X PBS

0.4 mL

0.8 mL

1.6 m

Distilled water

3.52 mL

7.04 mL

14.08 mL

Phosphatase Inhibitors

40 µL

80 µL

160 µL

Deacetylase Inhibitor

40.0 µL

80.0 µL

160.0 µL

Total Required

4.0 mL

8.0 mL

16.0 mL

10X Hypotonic Buffer

25.0 µL

50.0 µL

100.0 µL

Distilled water

217.5 µL

435.0 µL

870.0 µL

Phosphatase Inhibitors

2.5 µL5.0 µL10.0 µL

Deacetylase Inhibitor

2.5 µL5.0 µL10.0 µL

Protease Inhibitor Cocktail

1.25 µL

2.5 µL5.0 µL

100 mM PMSF

1.25 µL

2.5 µL5.0 µL

Total Required

250.0 µL

500.0 µL

1.0 mL

Digestion Buffer

48.5 µL

97.0 µL

194.0 µL

Phosphatase Inhibitors

0.5 µL1.0 µL2.0 µL

Deacetylase Inhibitor

0.5 µL1.0 µL2.0 µL

Protease Inhibitor Cocktail

0.25 µL

0.5 µL1.0 µL

100 mM PMSF

0.25 µL

0.5 µL1.0 µL

Total Required

50.0 µL

100.0 µL

200.0 µL

Complete
Hypotonic Buffer

Complete
Digestion Buffer

Vortex/mix thoroughly and keep the solutions on ice.

Nuclear Extraction from Cells
The following protocol is based samples of approximately 8.8 x 106 cells, which corresponds
to HeLa cells grown to confluence in a 100 mm tissue culture plate. Each sample is one
extraction reaction. Prepare PBS/Inhibitors, Complete Hypotonic and Complete Digestion
Buffers as described above. Adjust the volumes accordingly using the chart above if using
plates of different sizes. Place buffers and any tubes needed on ice before beginning.
Step 1: Cell Collection
1.

After growing and inducing/treating your cells as desired, aspirate the media out ofthe plate. Wash with 5 mL ice-cold PBS/Inhibitors. Aspirate solution out, then add 3mL ice-cold PBS/Inhibitors.

2.

Remove cells from dish by gently scraping with cell lifter. Transfer cells to a pre-chilled
15 mL conical tube.

3.

Centrifuge cell suspension for 5 minutes at 1500 rpm in a centrifuge pre-cooled
at 4ºC.

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5

4.

4.Discard supernatant. Keep cell pellet on ice. At this point the protocol can becontinued or the pellet can be frozen at -80°C.

Step 2: Isolation of the Nuclei
1.

Gently resuspend cell pellet in 500 µL freshly prepared (same day) Complete
Hypotonic Buffer by pipetting up and down several times. Transfer to a pre-chilled
microcentrifuge tube. Incubate on ice for 15 minutes.

2.

Add 25 µL Detergent and gently pipet up and down 3-5 times to mix.

3.

Centrifuge suspension for 30 seconds at 14,000 x g in a microcentrifuge pre-cooled
at 4ºC.

4.

Discard supernatant (cytoplasmic fraction). Keep the pellet (nuclear fraction) on ice.

Step 3: Nuclear Fraction Digestion and Collection
1.

Resuspend nuclear pellet in 100 µL freshly prepared (same day) Complete Digestion
Buffer by pipetting up and down 3-5 times.

2.

Add 0.5 µL of the Enzymatic Shearing Cocktail. Vortex gently for 2 seconds.

3.

Incubate suspension for 10 minutes at 37°C water bath. Vortex the sample gently
every 2-3 minutes during this incubation step.

4.

Add 2 µL 0.5 M EDTA to stop the reaction. Vortex gently on a low setting for 2
seconds and place on ice for 5 minutes.

5.

Centrifuge for 10 minutes at 14,000 x g in a microcentrifuge pre-cooled at 4ºC.
Transfer the supernatant into a pre-chilled microcentrifuge tube.

6.

Save a small aliquot for protein quantification by a Bradford-based assay. Aliquot andstore at -80ºC. Avoid freeze/thaw cycles.

Note:

The presence of certain detergents may interfere with protein determination
assays, such as Bradford or BCA. We therefore recommend using theComplete Digestion Buffer as the blank and performing a 1:50 or 1:250
dilution of your samples. Alternatively, try Active Motif’s ProStain™ Protein
Quantification Kit, which is ideal for quantifying extracts because it offers
greater sensitivity and resistance to many contaminating reagents used in
extract preparation.

Nuclear Extraction from Tissue
Note:

We recommend using only fresh tissue samples, as nuclear extraction withfrozen tissue is very inefficient. If you have frozen tissue, we suggest you use
the Whole-cell Extraction from Tissue protocol on page 10.

Step 1: Tissue Homogenization
1.

Weigh tissue and dice into very small pieces using a clean razor blade. Collect pieces
in a pre-chilled, clean Dounce homogenizer.

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6

2.

On ice, add 3 mL ice-cold Complete Hypotonic Buffer per gram of tissue andhomogenize in the Dounce. Incubate on ice for 15 minutes.

3.

Centrifuge for 10 minutes at 850 x g at 4°C.

4.

At this point, the tissue is homogenized. However, most of the cells are not yet lysed.
Therefore, discard the supernatant and continue with the pellet at Step 2, No. 1 of the
Nuclear Extraction Procedure from Cells on page 6, based on a 150 mm plate (2 x
107 cells).

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7

Protocols – Whole-cell Extraction
Buffer Preparation and Recommendations
Note:

Phosphatase Inhibitors, PMSF and PIC lose activity 24 hours after dilution.
Therefore, use all of the solutions below on the same day prepared, thendiscard any remaining solution.

Preparation of PBS/Inhibitors (8 mL/plate)
To make whole-cell extract from a 100 mm plate of cells, prepare 8 mL of PBS/ Inhibitors
solution as follows: mix 0.8 mL 10X PBS in 7.36 mL distilled water, then add 80 µl
Phosphatase Inhibitors and 80 µL Deacetylase Inhibitor. Vortex to mix and place on ice. (Thisbuffer is used for both whole-cell and nuclear extractions.)
Preparation of Complete Whole-cell Lysis Buffer (100 µL/plate)
To make whole-cell extract from a 100 mm plate of cells, prepare 100 µL Complete Wholecell Lysis Buffer as follows: mix 97 µL Whole-cell Lysis Buffer, 1 µL Phosphatase Inhibitors, 1µL Deacetylase Inhibitor, 0.5 µL Protease Inhibitor Cocktail (PIC) and 0.5 µL 100 mM PMSF.
Vortex to mix and place on ice.

Quick Chart for Preparing Whole-cell Extraction Buffers
60mm plate or 100mm plate or
3.2 x 106 cells
8.8 x 106 cells

150mm plateor 2 x 107 cells

Reagents to prepare

Components

PBS/Inhibitors

10X PBS

0.4 mL

0.8 mL

1.6 m

Distilled water

3.52 mL

7.04 mL

14.08 mL

Phosphatase Inhibitors

40.0 µL

80.0 µL

160.0 µL

Deacetylase Inhibitor

40.0 µL

80.0 µL

160.0 µL

Total Required

4.0 mL

8.0 mL

16.0 mL

Whole-cell Lysis Buffer

48.5 µL

97.0 µL

194.0 µL

Phosphatase Inhibitors

0.5 µL1.0 µL2.0 µL

Deacetylase Inhibitor

0.5 µL1.0 µL2.0 µL

Protease Inhibitor Cocktail

0.25 µL

0.5 µL1.0 µL

100 mM PMSF

0.25 µL

0.5 µL1.0 µL

Total Required

50.0 µL

100.0 µL

200.0 µL

Complete Whole-cell
Lysis Buffer

Vortex/mix thoroughly and keep the solutions on ice.

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8Whole-cell Extraction from Cells
The following protocol is based on samples of approximately 8.8 x 106 cells, whichcorresponds to HeLa cells grown to confluence in a 100 mm tissue culture plate, and will
yield approximately 0.20-0.35 mg of protein. Each sample is one extraction reaction. Prepare
PBS/Inhibitors and Complete Whole-cell Lysis Buffer as described above. Adjust volumes
accordingly using the chart above if using plates of different sizes. Place buffers and all tubes
needed on ice before beginning.
Step 1: Cell Collection
1.

After growing and inducing/treating your cells as desired, aspirate the media out ofthe plate. Wash with 5 mL ice-cold PBS/Inhibitors. Aspirate solution out, then add 3mL ice-cold PBS/Inhibitors.

2.

Remove cells from dish by gently scraping with cell lifter. Transfer cells to a pre-chilled
15 mL conical tube.

3.

Centrifuge cell suspension for 5 minutes at 1500 rpm in a centrifuge pre-cooled
at 4ºC.

4.

Discard supernatant. Keep cell pellet on ice. At this point the protocol can becontinued or the pellet can be frozen at -80°C.

Step 2: Whole-cell Extraction
1.

Resuspend the cell pellet in 100 µL freshly prepared (same day) Complete Whole-cell
Lysis Buffer and vortex vigorously for 10 seconds.

2.

Incubate on ice for 30 minutes, or on a rotating platform at 4°C for 30 minutes.

3.

Vortex vigorously for 10 seconds.

4.

Centrifuge for 10 minutes at full speed in a in a centrifuge pre-cooled at 4ºC. Transfer
the supernatant into a pre-chilled microcentrifuge tube.

5.

Save a small aliquot for protein quantification by a Bradford-based assay. Aliquot andstore at -80ºC. Avoid freeze/thaw cycles.

Note:

The presence of certain detergents may interfere with protein determination
assays, such as Bradford or BCA. We therefore recommend using theComplete Whole-cell Lysis Buffer as the blank and performing a 1:50 or 1:250
dilution of your samples. Alternatively, try Active Motif’s ProStain™ Protein
Quantification Kit, which is ideal for quantifying extracts because it offers
greater sensitivity and resistance to many contaminating reagents used in
extract preparation.

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9Whole-cell Extraction from Tissue
1.

Weigh tissue and dice into very small pieces using a clean razor blade. Collect pieces
in a pre-chilled 15 mL conical tube.

2.

On ice, disrupt and homogenize tissue in 3 mL ice-cold Complete Whole-cell Lysis
Buffer per gram of tissue with a pre-chilled, clean Dounce homogenizer or a Polytron
device. Maintain temperature at 4°C throughout all procedures. Incubate on ice for
30 minutes.

Note:

Frozen tissue can be sliced very thinly and thawed in this buffer prior tohomogenization with the Dounce. When using a mechanical homogenizer,
begin homogenization at slow speeds until the tissue is broken into smaller
pieces and then increase the speed to the maximum for 45-60 seconds. Avoid
the generation of excess heat or foam.

3.

Transfer to pre-chilled microcentrifuge tubes, centrifuge at 10,000 x g for 10 minutes
at 4°C.

4.

Transfer supernatants to new pre-chilled tubes and centrifuge again. Pool
supernatants in a single tube. The supernatant fluid is the whole-cell lysate.
Sometimes a longer centrifugation is necessary to obtain a clarified lysate.

5.

Save a small aliquot for protein quantification by a Bradford-based assay. Aliquot andstore at -80ºC. Avoid freeze/thaw cycles.

Note:

The presence of certain detergents may interfere with the Bradford or BCA
assay, thus use the Complete Whole-cell Lysis Buffer as the blank and perform
a 1:50 or 1:250 dilution of your samples. As an alternative, try Active Motif’s
ProStain™ Protein Quantification Kit, which offers greater sensitivity andresistance to many contaminating agentsactivemotif.com|

10

Protocols – Co-Immunoprecipitation
Buffer Preparation and Recommendations
Preparation of Complete Co-IP/Wash Buffer (500 µL/sample for Co-IP; 2 mL/sample
for washes)
Complete Co-IP/Wash Buffer is used for both the Co-IP and washing steps, and thissame buffer is used whether your sample is nuclear or whole-cell extract. Because the
Phosphatase Inhibitors, PMSF and PIC lose their activity 24 hours after dilution, you must
use this buffer on the same day it is made. Therefore, plan your experiment so that the
Co-IP incubation and wash steps can be performed on the same day. Use the chart below to
prepare sufficient Complete Co-IP/Wash Buffer for the number of samples being processed:
Reagents to prepare

Components

1 Co-IP

5 Co-IP’s

Complete Co-IP/Wash Buffer

Co-IP/Wash Buffer

2.425 mL12.125 mL

Phosphatase Inhibitors

25.0 µL

125.0 µL

Deacetylase Inhibitor

25.0 µL

125.0 µL

Protease Inhibitor Cocktail

12.5 µL

62.5 µL

100 mM PMSF

12.5 µL

62.5 µL

Total Required

2.5 mL

12.5 mL

Vortex to mix thoroughly and keep the Complete Co-IP/Wash Buffer on ice.
Increasing Stringency (Optional)
The Protein G Magnetic Beads included in the Universal Magnetic Co-IP Kit were chosenbecause they exhibit a very small degree of non-specific binding. This helps ensure very low
background, enabling the Complete Co-IP/Wash Buffer used in the kit to be of low stringency,
which better preserves fragile protein complexes.
Particular cell lines, stimulations or antibodies may result in higher background. If yourexperiment has more background than is optimal, increase the number of washes performed
from 4 to 6.
In extreme cases, increase the stringency of the Complete Co-IP/Wash Buffer used during
the Wash steps. Keep in mind, however, that this may disrupt the protein complexes, causing
loss of co-immunoprecipitated proteins. (Do not increase the stringency of this buffer for
the Co-IP.)
You can increase the stringency of the Complete Co-IP/Wash Buffer by adding 5 M NaCl (notsupplied) and/or additional Detergent (included). Add up to a maximum final concentrationof 150 mM NaCl and/or up to a maximum final concentration of 0.5% Detergent. Co-IP/Wash
Buffer as supplied contains 50 mM NaCl and 0.1% Detergent (IGEPAL CA-630, an NP-40
equivalent). We recommend that you begin at the lower end of the ranges and increase
concentrations as needed.

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11

Co-Immunoprecipitation Protocol
Important: Please read the previous section, Co-IP – Buffer Preparation andRecommendations before starting the assay. It is highly recommended that all Co-IP
incubation and wash steps be performed on the same day. When possible, all IP steps shouldbe performed on ice. The protocol below provides volumes sufficient for one IP experiment.
It is recommended that you perform, in addition to your samples, a Negative Control IP with
Rabbit or mouse IgG, (use the same species as that of the primary antibody you are using for
IP). This will help identify non-specific binding.
Step 1: Preparation of Antibody/Extract mixture
1.

Combine 100-500 µg nuclear or whole-cell extract and 1-5 µg antibody in a final
volume of 500 µL Complete Co-IP/Wash Buffer in a pre-chilled 1.5 mL microcentrifuge
tube. Keep on ice.

2.

Incubate for 1-4 hours at 4°C on a rotator or a rolling shaker (e.g. a Labquake from
Barnstead/Thermolyne with a tube holder for microcentrifuge tubes).

3.

Centrifuge for 30 seconds at 4000 rpm in a in a centrifuge pre-cooled at 4ºC tocollect liquid from the inside of the cap.

Step 2: Addition and Incubation of Magnetic Protein G Beads
Note:

Protein G Magnetic Beads are shipped on dry ice but should not be re-frozen.
Upon receipt, store the beads at 4ºC. The beads are ready to use following
complete resuspension to a homogeneous slurry. For best results, gently
shake and roll the tube. The beads settle quickly, thus resuspend justbefore pipetting. If performing a large number of reactions, you may need toresuspend the beads more than one time while adding them to your samples.

4.

Add 25 µL Protein G Magnetic Beads to each tube and incubate for 1 hour at 4°C on
a rotator.

5.

Centrifuge for 30 seconds at 4000 rpm in a in a centrifuge pre-cooled at 4ºC tocollect liquid from the inside of the cap.

Step 3: Washing the Magnetic Protein G Beads
See Appendix A: Use of Magnetic Beads and Included Bar Magnet on page 15 for more
complete information on washing the magnetic beads and assembling a magnetic standusing the Bar Magnet and Sticky Dots that are supplied in the kit.
6.

Place tube on magnetic stand to pellet beads on the side of the tube.

7.

Carefully remove and discard supernatant.

8.

Add 500 µL Complete Co-IP/Wash Buffer and resuspend pellet completely by
pipetting up and down several times. Take care that the beads are not clinging to thepipet tips after pipetting. You may need to move the tubes away from the magnet
before resuspension.

9.

Carefully remove and discard supernatant.

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1210. Add 500 µL of Complete Co-IP/Wash Buffer. Repeat this wash step for a totalof 4 washes. After final wash, remove as much supernatant as possible using a
P200 Pipetteman.
11. Resuspend each bead pellet in 20 µL of 2X Reducing Loading Buffer (130 mM Tris
pH 6.8, 4% SDS, 0.02% Bromophenol blue, 20% glycerol, 100 mM DTT). You canvortex the samples to mix thoroughly. Samples can be stored at -20°C, or you can run
Westerns immediately.

Western Blotting Protocol
For your convenience, a brief Western blotting protocol is provided below. When performing
Western blot, individual optimization of antigen and antibody quantities may be required inorder to detect the protein of interest, and you may have Western/blotting protocols thatare more appropriate for you antibodies. It is recommended that in addition to your samples(and the negative control IP you performed) that you run include an Input control lane that
contains 5 µg of the nuclear or whole-cell extract sample that was used for Co-IP. Resuspend
the 5 µg of extract in a final volume of 10 µL distilled water, then add 10 µL of 2X Reducing
Loading Buffer.
Electrophoresis
1.

Boil all samples at 95-100°C for 3-5 minutes. Centrifuge for 30 seconds at full speed.
Transfer to a fresh tube.

2.

Load 10 µL of each sample and a molecular weight marker onto an appropriate
SDS-PAGE gel and run until migration front reaches bottom of gel. (For weaker
interactions, it may be necessary to load all 20 µL of the sample in order to see theprotein band.)Transfer & Blocking
3.

Just before the end of electrophoresis, incubate a polyvinylidene difluoride (PVDF)
membrane in 100% methanol for 30 seconds. Rinse the membrane in distilledwater, then incubate it in Transfer Buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine,
15% methanol).

4.

Transfer protein to the PVDF membrane (1.5 hours at 30 V, 170 mA max) using
Transfer Buffer.

5.

Incubate membrane 30 seconds in 100% methanol, then wash in distilled water for
5 minutes.

6.

Incubate the membrane in PBS + 0.1% Tween 20 (PBST) + 3% dry, non-fat skim milk
powder for 1 hour at room temperature.

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Incubation with Primary & Secondary Antibodies
7.

Dilute the primary antibody (at the appropriate dilution) in PBST + 3% milk for 1.5-2hours at room temperature. Wash the membrane 2 times for 10 minutes each in PBST.

8.

Incubate membrane with HRP-conjugated secondary antibody for 1 hour at room
temperature. The dilution of the secondary antibody conjugate will vary according
to manufacturer’s specifications. Wash the membrane 3 times for 10 minutes each
in PBST.

Chemiluminescence Reaction
9.

Prepare and use the chemiluminescent substrate according to the
manufacturer’s instructions.

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Appendix
Section A. Use of Magnetic Beads and Included Bar Magnet
Caution: The included neodymium bar magnet is extremely powerful and is easily
broken if handled incorrectly.
1.

The magnet should be stored in the provided tube.

2.

Be careful when working near metal objects or surfaces. A free magnet will jump
great distances onto nearby metal objects with surprising speed/force. This can breakthe magnet.

3.

If the magnet becomes attached to a flat metal surface, it should be removed bysliding it off the edge of surface. The magnet may be broken if you attempt to pull
one end away from the metal.

Assembly of Magnetic Stands
The provided Mini Glue Dots can be used to attach the bar magnet to an empty tip box tocreate an effective magnet stand. Commercially available magnetic stands can also be used.
Creating a magnetic stand for microcentrifuge tubes:
1.

Remove the covering tape from one side of two glue dots.

2.

Place two microcentrifuge tubes in the wells of an empty tip box (1000 µL) and place
the magnet directly against the tubes. This is the way the magnet will be positioned
when the glue dots are used to affix it to the box.

3.

Attach the glue dots on the bar magnet (the uncovered face of the dot is placed on
the magnet) as shown below.

4.

Remove the covering tape from the exposed side of the glue dot. Fix the magnet to
the tip box so that it is against the tubes. The magnetic stand is now ready for use.

Note:

The microcentrifuge tubes are held less securely in this assembled tube standthan in a typical commercially available magnetic stand. This is not a problem
if the below washing protocol is followed. That is, work with 1 tube at a time,and keep the tubes in the standard tube rack unless you are holding the tube
next to the magnet.

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Washing the Protein G Magnetic Beads
Washing is best performed one tube at a time, and should be performed as follows:
1.

Place the tube in a standard microcentrifuge tube rack and open the cap.

2.

Place the opened tube in the assembled magnetic stand. The beads will pellet
more rapidly if the bottom of the tube is held against the magnet, as shown below,
and then slowly lowered into the well. This will pellet the beads up onto the side of
the tube.

3.

Allow the beads to pellet completely and remove supernatant with a 1000 µL
pipetteman. You can either leave the tube in the rack or pull it out when you remove
the buffer. The beads will remain on the side of the tube, even when not next tothe magnet.

4.

Return the tube to the standard microcentrifuge tube back, add 500 µL Complete
Co-IP/Wash Buffer and resuspend pellet completely by pipetting up and down
several times.

5.

Repeat steps 2-4 until desired washing steps are complete. After the final wash
has been removed, the last traces of wash buffer should be removed with a 200µL pipetteman.

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16Section B. Troubleshooting Guide
Problem / Question

Possible cause

Recommendation

No or low signalof IP’d proteins byWestern blot

The level of expression of theproteins of interest are very low.

Confirm presence of proteins of interestin the extract. Alternatively, use more
extract in the IP reaction (up to 500 µg
per IP).

Target proteins lost or destroyed
during sample preparation.

Confirm presence of proteins of interestin the extract.

The protein/protein interaction ofinterest is dependent on specificinduction conditions.

Optimize conditions of culture
and induction.

The protein/protein interaction ofinterest is not stable and dissociates
during the extraction process.

Perform the Enzymatic Shearing Cocktail
incubation (Step 3, No. 3) for 90 minutes
at 4°C instead of 10 minutes at 37°C.

Amount of antibody used in theassay is too low.

Use more antibody in IP and/or WB;
perform a titration to determine
how much.

Antibodies used in IP and/or WB are
not suitable for these applications.

It is important to perform IP and WB
controls, as antibodies raised against
denatured antigen and native epitopes
may not be recognized. For an IP
Positive Control, use the same antigen ofinterest for IP and WB. For a WB Positive
Control, perform a direct WB analysis of
the extract.

Large number of competing proteinspresent in the sample.

Centrifuge the extract at 20,000 x g for
20 minutes before adding antibody. Thisshould remove insoluble aggregates.

Interfering substances present in
the extract.

Use the appropriate amount of inhibitors
when performing the assay.

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Problem / Question

Possible cause

Recommendation

Highbackground signal

Too much antibody used in IP
and/or WB.

Titrate antibody concentrations anddecrease primary antibody dilution or
incubation time.

Non-specific binding of otherproteins to the Magnetic Protein G
Beads or antibodies.

Preclear extracts with Magnetic Protein
G Beads prior to addition of the
primary antibody.

Wash steps are notsufficiently stringent.

See Increasing stringency (optional) on
page 11 for details.

Aggregated proteins in extracts.

Spin extracts at 20,000 x g for 20
minutes and discard pellet prior toaddition of antibody.

IgG heavy chains appear at 55 kDa,
light chains appear at 28 kDa.

Use different antibody origins for IP
and WB.

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18

Technical Services

China

Japan

EuropeNorth America

If you need assistance at any time, please call or send an e-mail to Active Motif Technical
Service at one of the locations listed below.

Toll free:877 222 9543

Direct:

760 431 1263

Fax:

760 431 1351

Email:

tech_service@activemotif.com

Direct:+32 (0)2 653 0001

Fax:

+32 (0)2 653 0050

Email:

eurotech@activemotif.com

Direct:+81 (0)3 5225 3638

Fax:

+81 (0)3 5261 8733

Email:

japantech@activemotif.com

Direct:(86)-21-20926090

Cell Phone:
Email:

18521362870
techchina@activemotif.com

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