Immunoprecipitation (IP) Protocol

--Protein sample

--Antibody specific to target protein

--Protein A or G agarose beads

--Lysis buffer (containing protease inhibitors)

--Wash buffer (containing protease inhibitors)

--Elution buffer

Protocol:

1) Wash cultured cells with pre-chilled PBS for 2 times carefully

2) Add in cold RIPA lysis buffer

3) Scrap cells off to clean 1.5ml eppendorf tubes with a clean, cold scraper. Put them on a low-speed rotating shaker for 15 min at 4°C

4) Centrifuge at 14,000 g 4°C for 15min, then transfer the supernatant to new tubes immediately

5) Wash protein A/G-agarose beads for 2 times with PBS and make a 50% protein A/G agarose working solution (in PBS)

6) Add in 50% protein A/G agarose with ratio of 100μl for a 1ml sample solution. Shake on horizontal shaker for 10minat 4°C (This step aims to eliminate non-specific binding proteins)

7) Centrifuge 14,000g at 4°C for 15min, then transfer the supernatant to new tubes and discard protein A/G-agraose beads

8) Quantify total protein with BCA assay or other methods

9) Dilute the total protein to 1μg/μl with PBS to decline the concentrations of detergents. If you feel the concentration of your target protein is low, you can dilute the total protein to 10μg/μl. (if it’s high enough)

10) Add in appropriate amount of primary antibody to approximately 500μl total volume

11) Slowly shake antigen-antibody complex on rotating shaker at 4°C overnight

12) Centrifuge 14,000g for 5s, and keep the pellet and wash with pre-chilled washing buffer (or cold PBS) for 3 times (800μl each)

13) Collect the supernatant to proceed to SDS-PAGE, western-blot, or mass spectra analysis