Sepharose preincubation

If an antipeptide antibody corresponding to the sequence around a candidate phosphorylation site is available, this antibody can be used for immunoprecipitation of proteolytic digests. To do this, preincubate the antibodies with protein A-Sepharose for 1 hr at 4°C. Collect the beads by centrifuging briefly in a microcentrifuge. Wash the beads several times with 50 mM... 

The supernatant is preincubated with 200 fiM GDP or GTP7S for 30 min at room temperature, then with 10 /xg of the GST-fusion protein prebound to glutathione-Sepharose beads for 1 hr at 4° with gentle shaking, and centrifuged at 2000g for 5 min at 4°. 

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