(no subject)

padma reddy reddypvr at BU.EDU
Tue Jul 22 11:35:00 EST 1997


Here are a couple of suggestions
(1) I pre-bind my antibody to Protein A Sepharose - the way I do it is
to prepare a mixture of Protein A sepharose (final 25%) , BSA (final 1
mg/ml) and the antibody ( final 3microgm /100 microlitres) in 10 mM
Tris-HCl, pH 7.5 and 150mM NaCl - incubate at room temp for 1 hr on a
rotatorque. Unbound antibody is washed off at the end of incubation with
the above buffer 3 times. Resuspend in teh same volume and use 100
microlitres for every immunoprecipitation either overnight for 4 hrs min
in teh coldroom.
(2) I do the washes in teh same buffer 2 time and the final wash in 10
mM tris-HCl, pH 7.5.
Hope you find them useful. Good Luck
Padma




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