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Summary: 84
Protein Immunoprecipitation
Binding Antibody to Protein-G Sepharose
Antibody Type Volume Sepharose (volume of 50% slurry)
mAb 2 - 4 µl ascites 30 µl Protein G
pAb 1 - 2 µl serum 30 µl 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 4°C.
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 4°C for 30
minutes. This will yield ~75% lysis of cells.
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