Immunoprecipitation Experimental Design Tips

Immunoprecipitation (IP) is a technique used to enrich a specific protein from a heterogeneous cell or tissue extract using a target-specific antibody. Co-immunoprecipitation (co-IP) is the pull-down of intact protein complexes. IP and co-IP are valuable and widely used techniques to identify protein-protein interactions and novel members of protein complexes. While the technique of IP is relatively simple, there are many variables involved and the inclusion of the proper experimental controls is vital for the optimization and accurate interpretation of your IP results.

Input Control

A whole lysate control should be included to ensure that the western blot portion of the experiment is working properly. If the target signal is seen in the whole lysate control but not in the IP sample, this shows that the antibody is working properly, however, it is likely that the IP enrichment failed.

Isotype Control

An isotype control is a necessary negative control in your experiment. The control isotype used in an IP experiment should match the IgG subclass of the primary antibody. Rabbits only have one IgG subclass, therefore choosing an isotype control is relatively simple. We recommend using Normal Rabbit IgG #2729 for experiments with rabbit polyclonal antibodies and Rabbit (DA1E) mAb IgG XP^® Isotype Control #3900 for experiments using monoclonal rabbit antibodies.

When using a primary antibody produced in mice, choosing an isotype control is more complicated, as mice have five IgG subclasses: IgG1, IgG2a, IgG2b, IgG2c, IgG3. The subclass of a specific CST antibody can be found on the product webpage in the Source/Isotype section. CST offers the following isotype controls to match our mouse primary antibodies:

• Mouse (G3A1) mAb IgG1 Isotype Control #5415
• Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656
• Mouse (E7Q5L) mAb IgG2b Isotype Control #53484
• Mouse (E1D5H) mAb IgG3 Isotype Control #37988

Isotype controls should be concentration matched and run alongside the primary antibody samples during western blotting.

Bead-Only Control

A bead-only control acts as an additional negative control for your beads within an IP experiment and involves adding beads to your lysate sample without any antibody present. It may be necessary to add a bead-only control if you are experiencing non-specific binding in your IgG isotype negative control and/or your IP experiment.

CST offers the following bead types for IP experiments:

• Protein A Agarose Beads #9863
• Protein G Agarose Beads #37478
• Protein A Magnetic Beads #73778
• Protein G Magnetic Beads #70024

Experimental Design

We recommend setting up the experiment as follows:

1. Prepare a tube for each of the 4 following conditions: Input, Isotype Control (concentration matched), Bead-Only Control, and Antibody (at the recommended dilution).
2. Add 50 µl of lysate to the "Input" tube and add 200 µl of lysate to each of the other tubes. Keep the "Input" tube on ice and follow our recommended IP protocol for the other tubes (i.e., Isotype Control, Bead-Only Control, and Antibody).
3. Add 25 µl 3X reducing SDS loading buffer to the "Input" tube and elute the remaining samples by re-suspending the bead pellets in 30 µl 3X reducing SDS sample buffer.
4. Heat samples (including "Input") to 95-100 C for 2-5 min. Microcentrifuge for 1 min at 14,000 x g and save IP eluates.
5. Load 15 µl of each input sample and IP eluate sample(s) on a gel and analyze it by western blot.

If your IP experiment does not end up going as planned, please visit the Immunoprecipitation Troubleshooting Guide for additional support or contact Technical Support.