Co-immunoprecipitation Guide

Introduction

Co-immunoprecipitation (Co-IP) is a widely used technique that leverages the specific binding between antigens and antibodies to study protein-protein interactions. It is commonly used to determine whether two target proteins interact to form a complex within an in vivo environment. Additionally, Co-IP can be used to identify potential binding partners of a specific protein.

Principle

When cells are lysed under non-denaturing conditions, intracellular protein–protein interactions can be preserved. An antibody specific to protein M is added to the cell lysate and incubated to allow binding. Pre-treated Protein A/G agarose beads are then introduced to capture the antibody-protein complex. If protein N interacts with protein M in the cell, a complex is formed in the following order: “protein N-protein M-anti-protein M antibody-Protein A/G-agarose beads.” The eluent can then be analyzed by SDS-PAGE, Western Blot, or mass spectrometry to confirm the interaction between the two proteins.

Method of Application

1. Preparation of target protein sample

1) Treatment of serum and secreted target protein samples: Collect serum or supernatant of culture medium, and detect the concentration of target protein. If the target protein concentration is high, it is recommended to dilute with 1×PBS until the final protein concentration is 10-100 μg/mL for subsequent experiments.

2) Preparation of cell lysate

     a. Collecting cells Blow the suspended cells and semi-adherent cells off the cell culture flask and transfer them into a centrifuge tube, centrifuge at 1,000 rpm for 5 min, and discard the supernatant. Gently scrape the adherent cells off the flask wall with a cell scraper, transfer them into a centrifuge tube together with the culture medium, centrifuge at 1,000 rpm for 5 min, and discard the supernatant.

     b. Re-suspend the cells with 1×PBS pre-cooled to 4 ℃, centrifuge at 1,000 rpm for 3 min, and discard the supernatant. Repeat.

     c. Add the corresponding volume of cell lysate according to the amount of cells, and place it on the ice for 10-20 min after repeated pipetting.

     Note: Generally, 1mL of cell lysis buffer can process about 0.5-1.0×10⁷ cells. To avoid degradation of the target protein, you may add protease inhibitor.

     d. Treat cell lysate with ultrasonic crusher until cell lysate is clear and no longer viscous. After 30 min on ice, centrifuge at 12,000 rpm and 4℃ for 10 min. The supernatant is taken as the protein sample. It is recommended to proceed to the next step of the experiment immediately. If time does not allow, store the protein sample at -80 ℃。

2. Binding of the antibody to the gel comple

1) Protein A/G gel preparation: Fully suspend the gel, take 40 μL gel suspension (containing 10 μL gel, the rest is the suspension liquid) in a centrifuge tube, add 250 μL 1×PBS, fully suspend, centrifuge at 1,000 rpm and 4℃ for 30 s, and discard the supernatant; Repeat this washing step twice.

2) Antibody preparation: According to the IP dilution ratio recommended in the antibody manual, dilute the antibody with 1×PBS to prepare an antibody working solution, or adjust the total volume of antibody to 500 μL. Keep it on ice for later use.

3) Add the diluted antibody to the pre-washed gel, mix it gently and incubate it at room temperature for 30 min on the shaking table.

4) Centrifuge at 1,000 rpm and 4℃ for 30 s , and take the supernatant into a new centrifuge tube for subsequent use.

5) Add 250 μL 1×PBS to the gel, mix gently, wash the gel, centrifuge at 1,000 rpm and 4℃ for 30 s, and discard the supernatant. Repeat this step four times. Get antibody gel complex.

3. Binding of target protein to the antibody gel complex

1) Incubation: Add 200 μL prepared sample to the antibody gel complex and incubate at room temperature for 30 min on the shaking table, or at 4℃ for 2 h or longer.

2) Centrifuge: After incubation, centrifuge at 1,000 rpm and 4℃ for 30 s, and discard the supernatant. Add 250 μL 1×PBST, mix gently, clean the gel, centrifuge at 1,000 rpm and 4℃ for 30 s, and discard the supernatant. Repeat four times.

4. Target protein elution and check

This manual provides the following two target protein elution schemes. Please select the appropriate target protein elution method according to the needs of subsequent detection.

 1) Denaturation elution method: It is applicable to SDS-PAGE, Western Blot detection.

     a. Add 2 μL 5×loading buffer into the gel complex, mix evenly, and boil the sample at 95℃ for 5 min.

     b. Centrifuge the gel, collect the supernatant, and perform detection.

Reference for experimental groups:

Input sample group: Total protein lysate (without immunoprecipitation).

IgG control group:Use the same-species IgG in place of the primary antibody.

Experimental group: Protein samples obtained after immunoprecipitation.

2) Acid elution method: It can be used for later functional analysis.

     a. Procedure: Add 100-200 μL acid eluent to the gel, incubate at room temperature for 10 min.

     Note: Acidic conditions can shorten the lifespan of the immunogel. Minimize the contact time between the gel and the acidic elution buffer, and it is recommended that this contact time not exceed 10 minutes.

     b. Centrifuge at 1,000 rpm and 4℃ for 30 s, collect the supernatant into the new collection pipe, and immediately add 10×PBS equal to 1/10th of the total volume of the supernatant for neutralization, adjust the pH of the eluted product to neutral, and the sample can be used for later functional analysis.

Reference for experimental groups:

IgG control group: Use the same-species IgG in place of the primary antibody.

Experimental group: Protein samples obtained after immunoprecipitation.