Enabling Epigenetics Research

Nuclear Complex Co-IP Kit
Catalog No. 54001(Version B3)

Copyright 2014 Active Motif, Inc.

Revision

Date

Description of Change

B3

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
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
Kit Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Kit Components and Storage  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Protocols  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
Nuclear Extraction – Buffer Preparation and Recommendations  . . . . . . . . . . . . . .  5
Quick Chart for Preparing Buffers  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Co-IP – Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Preparation of IP Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Co-Immunoprecipitation Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  9
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section A. Protein G Agarose Columns Wash Stand . . . . . . . . . . . . . . . . . . . . . . . . 13
Section B. Troubleshooting Guide  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

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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 and also
co-precipitate any bound interacting proteins within a sample. These protein complexes
are then captured by antibody binding beads (such as Protein A or G). This is followed byelution of the antibody-bound proteins, SDS-PAGE and Western blot using a second antibody
targeted against one of the bound, interacting proteins. 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. In addition, many unstable protein
complexes can be affected by the salt and detergent composition of the buffers used in the
immunoprecipitation process.
Because of this, Active Motif’s Nuclear Complex Co-IP Kit has been developed specificallyfor the preparation and immunoprecipitation of nuclear protein complexes from cell or tissue
samples. The kit is composed of two optimized modules: nuclear extract preparation and
Co-IP. The kit’s extraction module provides a simple, fast and effective method to obtain andmaintain protein complexes contained in nuclear compartments of the cell, specifically thosepreviously bound to DNA. The kit’s versatile Co-IP module enables the user to study manydifferent protein/protein interactions of varying strength.
In the kit’s extraction process the cells are collected in ice-cold PBS in the presence ofPhosphatase Inhibitors, which limits further protein modifications (expression, proteolysis,
dephosphorylation, etc.). The cells are then resuspended in Hypotonic Buffer to swell thecell membrane and make it fragile. Addition of Detergent causes leakage of the cytoplasmic
proteins into the supernatant. After removal of the cytoplasmic fraction, the nuclei are lysed
and the nuclear proteins are recovered in a low-salt buffer in the presence of the Protease
Inhibitor Cocktail and PMSF. This is followed by the addition of a proprietary Enzymatic
Shearing Cocktail. The use of low-salt buffers protects protein complexes in the nucleus; DNAdigestion allows a gentle release of undissociated protein complexes from the DNA. Afterthe protein complexes are collected, an immunoprecipitation reaction is carried out to detect
the bound proteins. In the immunoprecipitation module, two different immunoprecipitationbuffers with either a low or high stringency starting composition are provided. In addition,
detergent and salt are provided separately to enable the user to vary the salt and detergent
concentrations. The addition of salt and detergent is ideal for use with robust protein/protein
interactions because such conditions reduce background. However, as unstable protein
complexes may not withstand high stringencies, the kit format enables stringency to bemodified as required by each complex.
The Nuclear Complex Co-IP Kit provides sufficient reagents to perform 50 Co-IP experiments.
It includes optimized nuclear protein complex extraction reagents, immunoprecipitationbuffers and a comprehensive protocol to provide a simplified method to capture and detect
protein complexes.
Product

FormatCatalog No.

Nuclear Complex Co-IP Kit

50 rxns

54001

Protein G Agarose Columns

30 rxns53039

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1Flow Chart of Process������������������

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2

Kit Performance

250
1

2

3

Figure 1: Co-IP of the p33 subunit of the RNA pol II complex.
HeLa cells were grown to confluence on 100 mm plates and nuclear extracts were prepared using
the kit’s extraction reagents. For IP experiments, the stringency of the IP High Buffer was increased
by supplementing with NaCl (150 mM Final) and Detergent (1% Final). 100 µg of nuclear extract
was used per IP reaction and incubated with either 2 µg p33 antibody or no antibody (negative
control). Protein G beads were added to each IP reaction. Following the IP, 2X sample buffer wasadded to each IP reaction, samples were boiled and run on an SDS-PAGE gel. Western blot analysis
was performed using RNA pol II mouse mAb at 0.1 µg/mL followed by anti-mouse HRP at 1:1000.
Detection of the p33/RNA pol II complex by the RNA pol II antibody (lane 3) demonstrates that the
Co-IP was successful in maintaining the protein complex. The input HeLa extract (lane 1) was run asa control for the western blot using 0.1 µg/mL RNA pol II.
Lane 1: Western blot control – 10 µg HeLa extract
Lane 2: Negative Control – no antibody added in IP
Lane 3: Co-IP: IP p33/WB RNA pol II

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3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 Hypotonic Buffer

50 mL

4°C for 6 months

Phosphatase Inhibitors

9 mL-20°C for 6 months

Protease Inhibitor Cocktail

626 µL-20°C for 6 months

10X PBS

80 mL

4°C for 6 months

Detergent

22.8 mL

RT for 6 months

100 mM PMSF

50 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

5X IP Low Buffer

22.5 mL

4°C for 6 months

5X IP High Buffer

22.5 mL

4°C for 6 months

Dithiothreitol (DTT)

225 µL-20°C for 6 months

5 M NaCl

6.8 mLRT for 6 months

BSA

300 mg4°C for 6 month

Additional Materials Required• 5 and 10 mL pipettes• 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)• Protein G Agarose Columns (Active Motif Catalog No. 53039) or Antibody Binding Beads(such as Protein A or G beads)• Antibodies for IP and Western blot
• 37°C Water Bath• Reagents and equipment for performing SDS-PAGE and Western blot

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4

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 whole-cell extract from cells or from tissue) to determine
the updated amount of PBS/Phosphatase Inhibitors solution that will be required foryour extraction(s).
Preparation of PBS/Phosphatase Inhibitors
The Phosphatase Inhibitors solution should be a clear yellow color. It may precipitate duringstorage and become turbid in appearance. If this occurs, heat at 50°C for 10 minutes. To
make nuclear extract from a 100 mm plate of cells, prepare 8 mL of PBS/Phosphatase
Inhibitors solution as follows: mix 0.8 mL 10X PBS in 7.120 mL distilled water, then add 80 µL
Phosphatase Inhibitors. The Phosphatase Inhibitors lose their activity 24 hours after dilution.
Therefore, use the PBS/Phosphatase Inhibitors solution the same day it is prepared anddiscard any remaining solution.
Preparation of 1X Hypotonic Buffer
To make nuclear extract from a 100 mm plate of cells, prepare 500 µL of 1X Hypotonic Buffer
as follows: mix 50 µL 10X Hypotonic Buffer and 450 µL distilled water. Any remaining 1X
Hypotonic Buffer can be stored at 4°C for 1 week.
Preparation of Complete Digestion Buffer
To make nuclear extract from a 100 mm plate of cells, prepare 100 µL of Complete Digestion
Buffer as follows: mix 98.5 µL Digestion Buffer with 1 µL Protease Inhibitor Cocktail (PIC)
and 0.5 µL PMSF. The PMSF and PIC lose their activity 24 hours after dilution. Therefore,
use the Complete Digestion Buffer solution the same day that it is prepared and discard any
remaining solution.

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5Quick Chart for Preparing 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/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

225.0 µL

450.0 µL

0.9 mL

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

1X Hypotonic Buffer

Complete
Digestion Buffer

Preparation of Nuclear Extract
Starting from Cells:
The following protocol is based on samples of approximately 8.8 x 106 cells, whichcorrespond 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/Phosphatase Inhibitors, Hypotonic Buffer and Complete Digestion Buffer as describedabove in the section Buffer Preparation. Adjust the volumes accordingly using the chart
above if using plates of different sizes. Place buffers and any tubes needed on ice before
beginning assay.
Section A: Cell Collection
1.

Aspirate media out of dish. Wash with 5 mL ice-cold PBS/Phosphatase Inhibitors.
Aspirate solution out then add 3 mL ice-cold PBS/Phosphatase 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 430 x g 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.

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6Section B: Isolation of the Nuclei
1.

Gently resuspend cell pellet in 500 µL 1X Hypotonic Buffer by pipetting up and down
several times. Transfer to a pre-chilled microcentrifuge tube. Incubate for 15 minutes
on ice.

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 pellet (nuclear fraction) on ice.

Section C: Nuclear Fraction Digestion and Collection
1.

Resuspend nuclear pellet in 100 µL Complete Digestion Buffer by pipetting up anddown 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.

Note:

If you are studying protein/protein interactions that may be fragile or unstable,
perform this Enzymatic Shearing Cocktail incubation at 4°C for 90 minutes.

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
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 the Complete
Digestion Buffer as the blank and performing a 1:50 or 1:250 dilution of yoursamples. Alternatively, try Active Motif’s ProStain™ Protein Quantification Kit,
which is ideal for quantifying extracts because it offers greater sensitivity andresistance to many contaminating reagents used in extract preparation.

Starting from Tissue:
Section D: Tissue Homogenization
1.

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

2.

On ice, add 3 mL ice-cold 1X Hypotonic Buffer supplemented with DTT and Detergent(3 µL of the provided 1 M DTT and 3 µL of the provided Detergent) per gram of tissue
and homogenize. 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, continue the procedure with the cell pellet in Nuclear Extract Section B,
No. 1 on page 7, based on a 150 mm plate (2 x 107 cells).
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7

Co-IP – Buffer Preparation and Recommendations
The Nuclear Complex Co-IP Kit has been designed to enable the study of a variety of protein/protein interactions. The Kit includes two IP Buffers with different stringencies: IP High has ahigher stringency than IP Low. Each IP Buffer can be further supplemented with the provided
salt and detergent depending on the complex being studied. Increased stringency is suitable
for stable protein complexes as it reduces background levels without disrupting protein
interactions. IP Buffers containing low salt and detergent enable the detection of more
weakly bound protein complexes. If the strength of the protein complex is not known, we
recommend performing a series of experiments to determine the optimal buffer stringency as
outlined in the table below.
Approach

IP Buffer

5 M NaCl

Detergent

5 M NaCl for
Wash Buffer

1. No supplements

1X High

N/A

N/A

N/A

1X Low

N/A

N/A

N/A

1X High

150 mM Final

N/A

N/A

1X Low

150 mM Final

N/A

N/A

1X High

150 mM Final

1% Final

N/A

1X Low

150 mM Final

1% Final

N/A

1X High

150 mM Final

1% Final

300 mM Final

1X Low

150 mM Final

1% Final

300 mM Final

2. Addition of salt
3. Addition of salt and detergent
4. Addition of salt and detergent, andextra salt for final Wash Step

Preparation of IP Buffer
The IP Buffers should be supplemented with fresh Protease Inhibitor Cocktail (PIC) and DTT
before use. However, if DTT is not compatible with the protein complex being studied, do not use.
Note:

IP Incubation Buffer should be prepared fresh on Day 1. IP Wash Buffer should
be prepared fresh on Day 2. If you wish to increase stringency of the IP
Buffers add NaCl and Detergent as listed below. If you do not wish to increasestringency simply add dH2O to recommended Total Volume.
Volume Required for 1 Co-IP

Volume Required for 5 Co-IP’s

Day 1: IP Incubation

Day 2: IP Wash

Day 1: IP Incubation

Day 2: IP Wash

5X IP Buffer

100 µL

800 µL500 µL

4000 µL

PIC

2.5 µL

8 µL

12.5 µL

40 µL

1 M DTT0.5 µL

4 µL

2.5 µL

20 µL

5 M NaCl*

7.5 µL60 µL

37.5 µL

300 µL

Detergent*

45 µL

360 µL225 µL

1800 µL

dH2O

X µL

X µL

X µL

X µL

500 µL

4000 µL

2500 µL

20 mL

Total Volume*Add these components to the IP Incubation and Wash Buffers only if you wish toincrease stringency

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8

Co-Immunoprecipitation Protocol
Important: Please read the previous section, Co-IP – Buffer Preparation andRecommendations before starting the assay. When possible, all IP steps shouldbe performed on ice. The protocol below provides volumes sufficient for one IP
experiment and can be scaled up as needed. We also recommend including one IP
negative control (no antibody added in the IP reaction).
Day 1: Preparation of Antibody/Extract mixture
1.

Prepare IP Incubation Buffer as described in the above section. Each IP reaction will
require 500 µL.

2.

or preparation of samples, we recommend using 100-500 µg of extract and 1-5 µg ofantibody diluted to 500 µL in IP Incubation Buffer in a 1.5 mL microcentrifuge tube.
Keep tubes on ice.

3.

Incubate each Antibody/Extract mixture overnight at 4°C on a rotator.

Day 2: Addition of Antibody binding beads and IP of the Proteins of Interest
Active Motif recommends the use of its Protein G Agarose Columns (Catalog No. 53039)
for the capture of the IP reaction. The Protein G agarose beads supplied in the column are
pre-washed and ready to use. If using an alternative source of Protein A or G beads, thebeads should be thoroughly washed before use. Follow the protocol for the antibody capture
method being used.
1.

Prepare IP Wash Buffer as described in the above section. Each IP reaction will
require 4 mL of the IP Wash Buffer. Keep the buffer on ice until ready to use.

2.

Prepare a 100 mg/mL BSA solution by dissolving the provided BSA in 2.8 mL dH2O
For each IP reaction, transfer 1.5 mL of IP Wash Buffer to a new tube. Supplementwith 1 mg/mL BSA by adding 15 µL BSA to 1.5 mL IP Wash Buffer. Keep the buffer on
ice until ready to use.

If using Protein G Column-based Capture1.

Add the IP reaction directly to the column. Cap the column tightly to ensure it
is sealed. If desired, use parafilm to seal the cap. (Do not remove the tab fromthe column.)

2.

Incubate antibody/bead complex for 1 hour at 4°C on a rotator.

3.

During the incubation, prepare a 2X Reducing Buffer (130 mM Tris pH 6.8, 4% SDS,
0.02% Bromophenol blue, 100 mM DTT).

4.

Following the incubation, spin the column at 1250 x g for 1 minute to collect liquidfrom inside of the cap.

5.

The columns are designed to fit within an empty 1 mL pipet tip box which will serve asa stand for wash steps (see Appendix A for set up). Remove the cap and the tab fromthe bottom of the column and place the columns in the empty tip box. Add 500 µL
ice-cold IP Wash Buffer supplemented with BSA to the column. Allow flow-through tooccur by gravity.

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9

6.

Repeat the wash step twice with 500 µL ice-cold IP Wash Buffer supplemented
with BSA.

7.

Add 500 µL ice-cold IP Wash Buffer (no BSA) to each column.

Note:

To increase the stringency of this wash step, add 22.5 µL 5 M NaCl to each
500 µL of IP Wash buffer to be used.

8.

Repeat the wash step two more times with 500 µL ice-cold IP Wash Buffer (no BSA).

9.

Transfer the column to a microcentrifuge tube and centrifuge at 1250 x g at 4°C for 3
minutes to remove residual IP Wash Buffer.

10. Transfer the column to a new microcentrifuge tube.
11. Add 25 µL of 2X Reducing Buffer to each column and let stand for 1 minute at room
temperature. Centrifuge at 1250 x g at room temperature for 3 minutes to elute the
IP reaction.
12. Add 5 µL of pure glycerol to each 25 µL elution. Pipet up and down to mix.
13. Boil samples at 95-100°C for 5 minutes. Allow sample to cool to room temperatureand quick spin to collect condensation. Load gel for electrophoresis and subsequentWestern blotting.
If using Protein A or G Bead Capture in Solution
1.

Antibody binding beads (Protein A or G) must be washed thoroughly before addition
to the Antibody/Extract mixture from Day 1. For each IP reaction, aliquot 50 µL of
resuspended beads to fresh 1.5 mL microcentrifuge tubes.

2.

Add 250 µL IP Wash Buffer to each tube and flick to mix. Centrifuge at 1430 x g for
30 seconds at 4°C. Remove supernatant. Repeat wash step twice and place beads
on ice.

3.

Add 40 µL ice-cold IP Wash Buffer to the pelleted beads (total volume will exceed
60 µL).

4.

Add 60 µL of the washed beads to each Antibody/Extract tube from Day 1.

5.

Incubate antibody/bead complex for 1 hour at 4°C on a rotator.

6.

During the incubation prepare a 2X Reducing Loading Buffer (130 mM Tris pH 6.8, 4%
SDS, 0.02% Bromophenol blue, 100 mM DTT, 20% glycerol)

7.

Following the incubation, centrifuge tubes at 1430 x g for 30 seconds at 4°C. Remove
and discard the supernatant.

8.

Add 500 µL ice-cold IP Wash Buffer supplemented with BSA to each tube. Centrifuge
at 1430 x g for 30 seconds at 4°C. Remove and discard the supernatant. Repeat wash
step twice with 500 µL ice-cold IP Wash Buffer supplemented with BSA. Do not vortex
or flick tubes to mix. Keep tubes on ice when possible.

9.

Add 500 µL ice-cold IP Wash Buffer (no BSA) to the pelleted beads. Centrifuge at
1430 x g for 30 seconds at 4°C. Remove supernatant. Repeat wash step twice with
500 µL ice-cold IP Wash Buffer (no BSA). Do not vortex or flick tubes to mix. Keeptubes on ice when possible.

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10Note:

To increase the stringency of this wash step, add 22.5 µL 5M NaCl to each
500 µL of IP Wash Buffer to be used.

10. Resuspend each bead pellet in 8 µL of 2X Reducing Loading Buffer and boil samples
at 95-100°C for 5 minutes.
11. Allow sample to cool to room temperature, spin briefly to collect condensation andapply to gel for electrophoresis and subsequent Western blotting.
Western Blotting Protocol
For your convenience, a 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.
Electrophoresis
1.

Load sample onto an appropriate SDS-PAGE gel and run until migration front reaches
bottom of gel.

Transfer
2.

Transfer protein to polyvinylidene difluoride (PVDF) membranes at 400 mA
for 90 minutes using Transfer Buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine,
15% methanol).

Blocking
3.

Block membranes by incubating overnight at 4°C with shaking in 5% Blotto (5%dry, non-fat skim milk powder in PBS (pH 7.4)). The addition of 0.1% Tween 20 to
all Blotto and PBS solutions may increase the antibody specificity and decrease
the background.

Incubation with Primary Antibody
4.

Rinse membranes with PBS (pH 7.4). Dilute the antibody (at the appropriate dilution) in
5% Blotto. Incubate for 1 hour at 37°C, 2 hours at RT (room temperature) or overnightat 4°C with agitation.

5.

Decant antibody solution. Wash the membrane 5 times for 5-10 minutes at RT
in PBS. Washing should be performed with vigorous agitation over a minimum
30-minute period.

Incubation with Secondary Antibody
6.

Incubate membranes with secondary HRP conjugate diluted in 5% Blotto for 1 hourat RT with gentle shaking. The dilution of the secondary antibody conjugate will vary
according to manufacturer’s specifications.

7.

Repeat step 5.

8.

Wash membrane with PBS for 5 minutes with agitation before proceeding to thechemiluminescence reaction.

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11Chemiluminescence Reaction
9.

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

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12

Appendix
Section A. Protein G Agarose Columns Wash StandThe Protein G Agarose columns are designed to fit within an empty 1 mL pipet tip box which
will serve as a stand for wash steps. Remove the cap and the tab from the bottom of thecolumn and place the columns in the empty tip box. All wash steps can be performed using
gravity flow.

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13Section B. Troubleshooting Guide
Problem/question

Possible Cause

Recommendation

No or low signalof IP’d proteins byWestern blot

Proteins of interest are not
located in the nucleus or theirlevel of expression is very low.

Confirm presence of proteins of interest in the
nucleus and nuclear 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 interest in the
nuclear extract.

The protein/protein interaction of Optimize conditions of culture and induction.
interest is dependent on specificinduction conditions.
The protein/protein interactionof interest is not stableand dissociates during theextraction process.

Perform the Enzymatic Shearing Cocktail
incubation 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 atitration to determine how much.

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

It is important to perform IP and WB controls, as
antibodies raised against denatured antigen andnative epitopes may not be recognized. IP Positive
Control - IP antigen of interest and WB same
antigen. WB Positive Control - direct WB analysis of
the extract.

The IP buffer is too stringent.

Use the IP High and IP Low buffers without theaddition of salt and detergent. Favor use of IP Low
buffer, which is less stringent.

Large number of competing
proteins present in the sample.

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

Interfering substances present in
the nuclear extract.

Use the appropriate amount of inhibitors when
performing the assay.

Antibody breakdown.

Reduce concentration of DTT, or avoid use.

Interference with theestablishment or maintenance of
the antigen-antibody complex.

Reduce concentration of detergent.

Incorrect bead typeused to precipitate theantigen-antibody complex.

Antibody binding beads are not included in the kit.
Confirm Protein A or G use.

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Problem/question

Possible Cause

Recommendation

Highbackground signal

Too much antibody used in IP
and/or WB.

Titrate antibody concentrations and decreaseprimary antibody dilution or incubation time.

Non-specific binding of otherproteins to the Protein A/Gbeads or antibodies.

Preclear extracts with Protein A/G beads prior toaddition of the primary antibody.

Wash steps are notsufficiently stringent.

0.5% sodium deoxycholate can be added to thelast 3 IP washes and may reduce signal from thenegative (no antibody) control.

Aggregated proteins in extracts.

Spin extracts at 20,000 x g for 20 minutes anddiscard pellet prior to addition of antibody.

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

Use different antibody origins for IP and WB.

Can whole-cell
extracts be used?

If you wish to work with whole-cell extracts, werecommend use of Active Motif’s
Universal Magnetic Co-IP Kit, which contains
reagents for making both nuclear or whole-cell
extracts. It also utilizes Protein G Magnetic Beads
and a magnet for the IP and Wash steps, which
greatly reduces background.

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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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