You are now leaving the BD Biosciences website. The site you are about to visit is operated by a third party. The link to this site neither makes nor implies any representation or warranty for any products or services offered on a third-party site and is intended only to enable convenient access to the third-party site and for no other purpose. Do you want to continue?
For the best web browsing experience, please use Chrome, Safari or Firefox, minimum versions 77.0.3865, 12.1.2 and 68, respectively.
Immunoprecipitation with Antibody: Agarose Conjugates
PREPARATION OF THE CELL LYSATE
- Rinse a 60 mm culture dish of confluent cells with 1X phosphate-buffered saline (PBS).
- Add 0.5 ml boiling lysis buffer (1% SDS, 1.0 mM sodium ortho-vanadate, 10 mM Tris pH 7.4) to the culture dishes.
- Scrape the cells from the dish, transfer lysate to a 1.5 ml microcentrifuge tube, and boil for 5 minutes in a boiling water bath.
- Pass several times through a 26 gauge needle or sonicate to reduce viscosity; centrifuge (16,000 x g) for 15 minutes. The supernatant is the "total cell lysate (denatured)".
- Rinse a 60 mm culture dish of confluent cells with PBS.
- Add 0.5 ml cold immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0.2 mM sodium ortho-vanadate, protease inhibitor cocktail (Boehringer Mannheim), 0.5% IGEPAL CA-630) to the dishes to lyse cells.
- Maintain constant agitation for 30 minutes at 4°C in order for complete lysis to occur.
- Scrape the cell from the dish and pass several times through a 26 gauge needle to disperse any large aggregates and reduce viscosity. Centrifuge (16,000 x g, 4°C) for 15 minutes; keep on ice. The supernatant is the "total cell lysate (native)".
It is important to pre-clear the lysate immediately before immunoprecipitation.
- To 750-1000 µl of supernatant (denatured lysate or native total lysate), add 5 µg of rabbit anti-mouse IgG antibody, vortex, then add 75-100 µl of Protein A:Agarose (Cat. No. 610437). Incubate at 4°C for 30 minutes with gentle agitation.
- Centrifuge lysate (9000 x g, 4°C) for 2 minutes to pellet the agarose beads. The supernatant is the "total cell lysate".
- To a microcentrifuge tube, add 25 µl of a 50% suspension of antibody:agarose conjugate, 400 µl of water, 200-500 µg of total cell lysate in 100 µl and 500 µl of 2X immunoprecipitation buffer (2% Triton X-100, 300 mM NaCl, 20 mM Tris pH 7.4, 2 mM EDTA, 2 mM EGTA pH 8.0, 0.4 mM sodium ortho-vanadate, protease inhibitor cocktail (Boehringer Mannheim), 1.0% IGEPAL CA-630).
- Vortex and incubate for one hour with agitation at 4°C.
- After incubation, centrifuge the tube at 8000 x g, 4°C for 2 to 5 minutes, then aspirate the supernatant carefully without disturbing the agarose pellet.
- Wash the agarose beads with cold 1X immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1mM EGTA pH 8.0, 0.2 mM sodium ortho-vanadate, protease inhibitor cocktail, 0.5% IGEPAL CA-630) by centrifuging 2 minutes (8000 x g, 4°C). Decant supernatant and repeat wash twice.
- Resuspend pellet in 50 µl of 0.1 M Glycine pH 2.5 vortex and incubate with agitation for 10 minutes at 4°C to elute the proteins from the capture beads.
- Centrifuge (9000 x g, 4°C) for 2 minutes. Collect the supernatant, this is your IP sample.
- Add 5 µl of 1 M Tris pH 8.0 to each tube to neutralize the pH. Add approximately 10 µl of 5X concentrated electrophoresis sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% ß-mercaptoethanol) to each sample, and boil for 5 minutes. Centrifuge at 8000 x g for 30 seconds to spin down aggregates.
- Load the supernatant onto an SDS-PAGE gel and electrophorese.
- Transfer to PVDF and probe with appropriate antibodies (Refer to Western blot protocols for monoclonal or polyclonal antibodies).
APO-BRDU and APO-DIRECT are trademarks of Phoenix Flow Systems.