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Protein Immunoprecipitation Binding Antibody to Protein-G Sepharose

Summary: 84
Protein Immunoprecipitation
Binding Antibody to Protein-G Sepharose
Antibody Type Volume Sepharose (volume of 50% slurry)
mAb 2 - 4 l ascites 30 l Protein G
pAb 1 - 2 l serum 30 l Protein A
1. Wash sepharose beads ("beads") 3 X with IP buffer + 0.1% NP-40 ("IP/NP40").
IP buffer: 50 mM Tris-HCl, pH 8, 150 mM NaCl.
Collect beads by microcentrifugation for 30 seconds at 2000 rpm. Carefully remove as much
supernatant as possible. Leave some supernatant behind to prevent aspiration of beads.
2. Resuspend beads in 1 ml of IP/NP40. Add monoclonal antibody. Rotate for 1 hour at 4C.
3. Wash beads with IP/NP40: 2 quick washes, then 3 X 5 minutes (on rotator). Store beads on ice
until needed.
Immunoprecipitation From Yeast Cells
1. Plan for ~5 OD units of cells per IP. Chill cells on ice. Wash cells with cold IP/NP40 + protease
inhibitor cocktails ("IP/NP40/PICs"). Washed cells may be frozen until use. If starting with
frozen pellet, thaw quickly by addition of buffer in step 2 and immediately go to step 3.
2. Resuspend up to 25 OD units in 0.5 ml IP/NP40/PICs + 0.1 mM DTT ("IP/NP40/PICs/DTT").
3. Add 0.25 g glass beads (0.5 mm) in 2 ml microfuge tube and place on vortex mixer at 4C for 30
minutes. This will yield ~75% lysis of cells.


Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida


Collections: Biology and Medicine