RIPA (RadioImmunoPrecipitation Assay) Lysis buffer:
Tris-HCl: 50 mM, pH 7.4 Nonidet P-40 (NP-40): 1%
NaCl: 120 mM
EDTA: 1 mM
PMSF: 1 mM
Leupeptin 1 μg/ml
Aprotinin 1 μg/ml
Na3VO4: 1 mM
Note: Ingredients labeled with * should be added right before each use.
PMSF degrades to a half after 30min in water.
Washing buffer :
lysis buffer:5M NaCl =100:1 (ensure NaCl not exceed 1M)
protein A/G-agarose beads
Specific antibody (MAb or PAb)
Carefully wash cultured cells with pre-chilled PBS for 2 times.
Add in cold RIPA lysis buffer (1ml for 107cells).
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.
Centrifuge at 14,000 g 4°C for 15min, transfer the supernatant to new tubes immediately.
Wash protein A/G-agarose beads for 2 times with PBS and make a 50% protein A/G agarose working solution (in PBS)
Add in 50% protein A/G agarose with ratio of 100μl for a 1ml sample solution. Shake on horizontal shaker for 10min, 4°C (This step aims to eliminate non-specific binding proteins)
Centrifuge 14,000g at 4°C for 15min, transfer the supernatant to new tubes and discard protein A/G-agraose beads Quantify total protein with BCA assay or other methods.
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)
Add in appropriate amount of primary antibody to approximately 500μl total volume..
Slowly shake antigen-antibody complex on rotating shaker at 4°C for overnight.
Note: if downstream experiment is enzyme activity assay for kinase or phosphatase, it's better to change step 11 to a 2h incubation at room temperature.
12. Centrifuge 14,000g for 5s, 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.
Note: This Co-IP protocol is to bind antibody to the Protein A/G-argarose beads and then mix with the antigen. It gives lesser yield than the other one and avoids the problem of co-elution of antibodies. If you want to yield high purity of target protein regardless of non-specific binding, you can mix antibody with protein sample prior to addition of Protein A/G-agarose beads, thus in the end the antibodies are also co-eluted with target protein and interference might occurs in western blot detection.