发信人: netabc (dye), 信区: Biology
标 题: Re: 弱弱地问 how to lyse cells for western
发信站: BBS 未名空间站 (Wed Feb 1 20:56:59 2006)


You are right, RIPA comes from "radio immuno-precipitation assay" It's
originally used for immuno-precipitation. It contains 0.1~0.5% SDS to
completely lyse cells.

Protease inhibitors are used most time to keep protein from degradation by
various protease. And keeping your sample at 4C also helps this when
processing samples

Non ionic detergents, like NP-40, Triton-100 is much weeker than ionic one,
they may not lyse cell compeltely. Ususally, 0.2% SDS is enough to break
nuclear.

1x loading buffer can be used directly. The problem is (1) high SDS is not
compatible with most protein quantitation assay (2) All genomic DNA and total
proteins are in your samples. This ususally means more non-specific bands.

Depend on where your proteins most likely stay, select proper detergent, salt
concentration or pH.

Also you can get plenty of info from Biorad, Sigma, or Pi