Guide to immunoprecipitation experiments

Principle
Co-immunoprecipitation is a method used to detect whether there is an interaction between two protein molecules by taking advantage of the specific binding of antigens and antibodies and the specific binding of protein A or G to the Fc fragment of antibodies. Antibodies against protein M are added to cell lysates, incubated, and then Sepharose beads pretreated with Protein A or G are added. If there is a protein N in the cell that binds to protein M, such a complex can be formed: “protein N-protein M-antibody against protein M-Protein A or G-Sepharose beads”. The results are analyzed by SDS-PAGE electrophoresis. The protein N obtained in this way is naturally bound to protein M in the cell, which is consistent with the actual situation in vivo, and the results obtained are highly reliable. This method is often used to determine whether two target proteins bind in vivo; it can also be used to identify new partners for a specific protein.

Step 1
1. Wash adherent cells twice with pre-cooled PBS solution (centrifuge at 800-1000 rpm for suspended cells).
2. Add pre-cooled RIPA buffer (with protease and phosphatase inhibitors). RIPA buffer usage: 0.5 mL per 5×106 cells.
Note: For suspension cells, collect the cell sediment and add pre-chilled RIPA buffer (containing PMSF). The amount of RIPA buffer used is calculated as 0.5 mL per 5 × 106 cells. Gently mix the cell suspension and use an oscillator to lyse the cells at 4°C for 15 min.
3. Transfer the adherent cells to a centrifuge tube using a rubber or plastic cell scraper pre-cooled with distilled water.
4. Centrifuge the lysate at 10,000 rpm for 15 min at 4°C, quickly transfer the supernatant to another centrifuge tube, and discard the precipitate.
5. Mix the Agarose protein A+G beads: Wash the Agarose protein A+G beads twice with pre-chilled PBS and resuspend them in PBS to a 50% suspension.
6. Add 100 L Agarose protein A+G beads to 1 mL of cell lysate supernatant and mix well. Pre-clear the mixture by shaking at 4 °C for 10 min.
7. Centrifuge at 10,000 rpm for 10 min at 4 °C to remove the Agarose protein A+G beads. Transfer the supernatant to a new centrifuge tube.
8. Determine the protein concentration in the supernatant by the Bradford method (Coomassie brilliant blue staining method). (The cell lysate should be diluted at least 1:10 before measurement, because the detergent components present in the lysate will interfere with the action of Coomassie brilliant blue).
9. Dilute the cell lysate protein concentration measured to 1 mg/mL with PBS to reduce the concentration of detergent in the buffer system (however, for those proteins that are expressed at low levels in cells, a higher concentration such as 10 mg/mL may be more effective for immunoprecipitation).
10. Add the recommended volume of immunoprecipitation antibody to 500 L of cell lysate to react with the target protein. (The optimal amount of antibody should be determined based on the amount of immunoprecipitated target protein in each cell model.)
11. Mix the cell lysate and antibody gently and incubate for 2 h, or overnight with slow shaking at 4 °C. (The choice of incubating for 2 h is commonly used for immunoprecipitation of activated enzymes that interact with kinases or phosphatases.) Add 100 L Agarose protein A+G and gently shake for 1 h or overnight at 4 °C to capture the immunoprecipitation complex.
12. Centrifuge (3000 rpm, 5 min) to collect the agarose beads, discard the supernatant, and wash the beads three times with 800 L-1000L pre-chilled PBS buffer. (Carefully aspirate the supernatant to avoid touching the bottom beads.)
13. Collect the precipitate, resuspend the precipitate in 60 L 2×SDS protein loading buffer, mix gently, boil for 5 min, and centrifuge at 12,000 rpm for 10 min.
14. Transfer the supernatant to a new centrifuge tube for Western blotting.

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