Co-Immunoprecipitation Guide

Principle

Co-Immunoprecipitation is a method to detect whether there is an interaction between two protein molecules, which is based on the specific binding of antigen and antibody. Firstly add the antibody specific to Protein M to cell lysis. After incubation, add Sepharose beads that are preprocessed by Protein A or Protein G. If there is Protein N that binds to Protein M in cells, then a kind of compound can be formed like this: “Protein N - Protein M - Anti-protein M antibody – Protein A/ Protein G-Sepharose beads”. The detection result can be analyzed by SDS-PAGE. The target Protein N obtained by the method above binds to Protein M naturally within cells, which accords with the actual in vivo situation and can obtain a high-reliability result. This method is often used to detect if two target proteins bind in vivo, and also used to confirm a new and uncertain protein that accompanies a specific protein.

Operation steps

1. Wash the adherent cells with pre-cooled PBS for 2 times. (Centrifugally wash the suspension cells at 800-1000 rpm.)

2. Add pre-cooled RIPA solution (containing PMSF), and the usage amount of RIPA solution can be calculated according to 0.5 mL/ 5×106 cells.

3. Transfer the adherent cells to a centrifuge tube with a plastic cell scraper that is pre-cooled with distilled water.

Attention: As for suspension cells, firstly collect the cells with a centrifuge, and then add pre-cooled RIPA solution which contains PMSF. The amount of RIPA solution can be calculated according to 0.5 mL/ 5×106 cells. Mix the cell suspension gently and homogeneously, and also use an oscillator to oscillate the cell suspension for 15 minutes to lyse the cells.

4. Centrifuge the lysate at 4℃ with 14000g for 15 minutes, then immediately transfer the supernatant to another centrifuge tube and discard the precipitate.

5. Prepare Protein A (or Protein G) - Sepharose: Wash Protein A (or Protein G) - Sepharose beads with PBS for 2 times, and make it into 50% suspension with PBS.

6. Add 100µL Protein A for each 1 mL supernatant of the cell lysis, and then oscillated at 4℃ for 10 minutes, finally pre-cleared. 

7. Separate Protein A (or Protein G)- Sepharose beads by centrifugation at 4℃ at 14000g, and then transfer the supernatant to a new centrifuge tube.

8. Use Bradford method (Coomassie brilliant blue staining) to determine the concentration of proteins in the supernatant. (The cell lysis should be diluted at least at 1:10 before the assay, because the detergent molecules that exist in the cell lysis would interfere in the reaction of Coomassie brilliant blue.)

9. According to the concentration of proteins in the cell lysis that is obtained from assay, dilute the proteins to 1mg/mL with PBS to reduce the concentration of detergent in the cell lysis. (But in terms of the proteins with lower levels of expression in cells, a higher concentration of 10mg/mL would be more effective for immunoprecipitation.)

10. Add the suggested volume of antibody for immunoprecipitation to 500µL cell lysis. (The optimum amount of antibody should be determined according to the quantity of target proteins in each cell model for immunoprecipitation.)

11. Mix the cell lysis and antibody gently and homogeneously, and then incubate it for 2 hours or oscillate it at 4℃ overnight. (The method of incubation for 2 hours is often applied to activating enzyme that reacts with kinase or phosphatase for immunoprecipitation.)  Then add 100 µL Protein A (or Protein G)-Sepharose, and gently oscillate for 1 hour or incubate at 4℃ overnight in order to obtain immunoprecipitation compounds.

12. Collect Sepharose beads by centrifugating at 14000 rpm for 5 seconds, and separate the supernatant. Then wash the beads with 800µL of the pre-cooled RIPA buffer for 3 times. 

13. Resuspend the Sepharose beads in 60 µL of 2×SDS Sample Loading Buffer, then boil the mixture solution for 5 minutes after gently homogeneous mixing, and separate the beads by centrifugating at 200g for 1 minute.

14. Transfer the supernatant to a new centrifuge tube to do the Western Blot assay.