General Protocol - Immunoprecipitation
Materials
Cell Lysis Buffer:
80 mM Na2HPO4
20 mM NaH2PO4
100 mM NaCl
0.1% SDS
1% Triton X-100
10% Glycerol
Filter and store at 4°C
Add the following ingredients just before use:
1 protease inhibitor cocktail tablet per 10 ml solution (Boehringer Mannheim, #1697498)
1 M DTT to final 1 mM (i.e. 10 µl per 10 ml solution)
6X Gel Loading Buffer:
375 mM TrisHCl, pH 6.8
45% Glycerol
0.5% Bromophenol Blue
10% SDS (better use powder)
15% ß-mercaptoethanol (add fresh if possible)
Lysate Preparation
- From cultured cells
1. Grow cells to about 80-90% confluence in 100-mm cell culture dish.
2. Leave the plate on ice for a few minutes.
*All subsequent procedures are performed on ice!!
3. Wash cells twice with cold PBS, aspirate any residual liquid.
4. Add 0.5~1 ml of cell lysis buffer per 100-mm culture dish. Make sure the entire surface is covered.
5. Leave the plate(s) on ice for 5 min. Swirl the plate(s) several times to ensure all cells are
lysed.
6. Scrape cells and collect the crude lysate in a pre-chilled 1.5 ml microcentrifuge tube.
7. Spin the lysate at maximum speed for 30 min at 4°C.
8. Collect the clear supernatant.
9. Measure the protein concentration by Bradford assay (BioRad, Cat. No. 500-0006). At this point the protein conc. is about 0.2-1 mg/ml.
10. Aliquot, snap freeze the lysate in N2(l) and store at -70°C. DO NOT freeze and thaw the lysate more than 2 more times.
Immunoprecipitation
1. Take a sample of cell lysate containing 10-100 ng target protein. Add 0.5-5 µl of polyclonal antiserum or 0.5-5 µg of monoclonal antibody. The amount of antibody to be added will depend on the amount of target protein to be precipitated, the volume of the reaction and the titer and avidity of the antibody. The antibody can be titrated in pilot experiments in which a fixed amount of antigen is precipitated by increasing amounts of antibody.
2. Incubate at 4°C for 1 h on a rocking platform.
3. If the anti-target antibody does not have a high affinity for protein A, add 0.5 µl of appropriate anti-immunoglobulin antibodies to the mixture after 30 min. Alternatively, protein G beads can be used instead or together with protein A beads.
4. Add 100 µl of protein A beads (10% vol/vol in lysis buffer) to the mixture. Incubate for 1 h at 4°C on a rocking platform.
5. Precipitate the beads by centrifugation at 10,000 g for 15 sec at 4°C. Remove the supernatant by gentle aspiration. Wash the immune complexes on the beads 3-5 times with lysis buffer. Each time add 1 ml of lysis buffer and resuspend the beads by vortexing. The final wash should be removed completely.
For SDS-polyacrylamide gel electrophoresis, add 30-50 µl of 1 x SDS gel-loading buffer (diluted from 6X). Denature the proteins in the sample by putting the tube in boiling water for 5 min. Centrifuge for 15 sec at 10000 g and load the supernatant on the gel.
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