Co-Immunoprecipitation

Co-Immunoprecipitation (Co-IP) is a powerful and popular method for analyzing protein-protein interactions by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein. These interacting proteins may be complex partners, structural proteins, co-factors, or signaling molecules, which are useful to help researchers to understand signaling transduction pathways.

Co-IP Service

Biologics International Corp. (BIC) provides custom co-IP service, in which we will design the optimal proposal based on the customer’ s specific needs. Using our mature recombinant expression platform, the technical group of BIC can also offer protein expression service when the target protein is identified. Contact us for project quotations and more detailed information.

Service	Customer Provides	Deliverables
Bait protein expression (4 weeks)	Customer provides gene sequence or plasmid of bait protein	Cloning vector, QC report, protein with the purity >90%
Co-IP (2 weeks)	You can provide the detection antibody >20 μL, or BIC can prepare the relevant antibody	Interaction analysis report, protocol report

The Principle of Co-IP

When cells are lysed under non-denaturing conditions, many intracellular protein-protein interactions can be retained. An antibody against a specific bait protein (such as protein X) is stabilized on the surface of agarose beads. If there is another protein (such as protein Y) can bind to protein X, the complex of protein X-protein Y will be precipitated together by the antibody, and any proteins not precipitated on the beads will be washed away. Subsequently, the interaction between protein X-protein Y can be confirmed by analyzing protein Y (usual methods of analysis are SDS-PAGE and western blot).

There are several advantages to the co-IP assay, including the following:

• Both the bait and prey proteins are in their native conformation.
• The interaction between protein happens in vivo with little to no external influence.
• In co-IP, the proteins interact under non-denaturing conditions which is almost physiological.

But this technology also has several limitations, including the following:

• Instability or transient interaction between proteins is not easily detected.
• The result of co-IP cannot determine whether the interaction is direct or indirect, since the breakage of cell membrane and organelles cause releasing of large of amount of proteins. Nonspecific interactions are inevitable.

In essence, co-IP is a more delicate version of a traditional immunoprecipitation. If you need other technologies to analyze the interaction between proteins or study binding kinetics, GST pull-down assay or biomolecular interaction analysis may be a good choice.

The Co-IP Protocol

Cell Lysis

The first step in co-IP cell lysis, which is an important step in co-IP assays. Cultured cells are washed twice with pre-chilled PBS, followed by addition of lysis buffer. After low-speed shaking for approximately 15 min at 4 °C, the cells lysate is centrifuged at 14,000 x g, 4 ℃ for 15 min.

Immunoprecipitation

Before this step, it may be necessary to perform a lysate pre-clearing step to reduce non-specific binding to the immunoprecipitation beads (usually protein A/G agarose beads that allowed purification of the antibody-protein complexes via centrifugation).

Primary antibody is added to the cell lysate supernatant, followed by incubation overnight at 4 ℃. Then, either protein A or G agarose beads are added, and are incubated with gentle rocking for 1-3 h at 4 ℃.

Elution of Target Protein

Most loading buffers of SDS-PAGE can reduce and denature proteins. According to the specific needs of the experiment, the elution methods can be classified as denaturing and non-denaturing.

• Denaturing elution

The beads are washed with lysis buffer or PBS for 3-5 times at 4 °C, followed by adding SDS loading buffer to resuspend. After microcentrifugation for 30 s, the sample is heated to 96 °C for 2-5 min and microcentrifuged for 1 min at 14,000 x g.

• Non-denaturing elution

Like denaturing elution, non-denaturing elution also requires washing of the beads and addition of 0.1 M glycine at a low pH (around 2.5-3) as a non-denaturing elution buffer. Be attention, as some proteins will be denatured or lose enzymatic activity under these conditions. Neutralization buffer can be added to the elution buffer to neutralize the pH.

Detection

The final step of co-IP is analysis of the target protein. Depending on the researcher’s needs, several methods (such as SDS-PAGE or native PAGE, western blot, mass spectrometry and enzymatic analysis) can be chosen.

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