Because both are detergents and are used in various biochemical assays. The precipitate formed due to TCA is soluble in standard sample solubilization buffer (8M Urea, 4% w/v CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-
propanesulfonate), 1% w/v DTT). Here can I use Triton-X-100 as a alternative to CHAPS? The soluble proteins are to be used in SDS-PAGE.
Hi Vishal,
why don't you just redissolve your proteins in SDS-PAGE sample buffer?
Achim
Hi Achim,
I tried to redissolve protein precipitate in SDS-PAGE sample buffer but it is not fully soluble in it. When i tried it to load on gel, it blocks wells of gel.
Hi Vishal,
I find this a bit surprising. There are only very few proteins that are not soluble when you boil them in SDS-PAGE sample buffer. Is it possible that the precipitate you are getting is not protein (could be e.g. a K+ SDS salt, which is not very soluble - Douglas Trask has exploited this for the development of a very nice method for finding proteins that bind tightly to DNA, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC557405/?page=1)?
I would try and take your TCA precipate, wash it very well with acetone to get rid of the TCA (if the bromophenol blue gets gree upon adding the SDS-PAGE sample buffer, the sample is still acidic and you don't want that), then either filter or spin, discard the precipitate and put the filtrate/supernatant onto the gel.
@ Achim Treumann
Thank you Sir,
I will definitely follow your suggestions.
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