Any good method for resuspension protein from TCA/acetone precip ? - (Dec/18/2007 )

Any good method for resuspension protein from TCA/acetone precip ?
I'll quantitate the protein after resuspension and then in-solution digestion
who can help me?




cheers

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For western analysis I resuspend whole cell lysates after TCA/acetone precipitation in 500mM Tris, 6.5% SDS, 100mM DTT, 9% glycerol (~5:1 sol'n"pellet). I lets these sit at 55 for 20-60min until chunks are easily resuspended in. Then I spin and mix 1:1 with LLB. I'm not sure how you want to quant or what you are digesting for but generally I only resuspend a TCA ppt for SDS-PAGE.

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i dry well the protein pellet and resuspend it in RIPA buffer 50° 30' to 1H on rotating heater.

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lowest amount of DMSO, some µl will do; after solving, try to add the buffer you need...

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Dear Bearer,
When you do a TCA precipitation ,Protein will lose its Tertiary structure and you wont get active protein after dissloving in solution of your interest. For quatnification of the protein , it is ok to dissolve in either of the buffers but for digestion your protien should be in active confromation(you wont get with TCA). AFter TCA you can dissolve the ppt in Electrophoresis sample buffer where you have SDS and 2BME. but for this also bromophenol blue will affect if you go for quantification, without bromophenol blue you can do estimation.

Correct me if iam wrong

a waiting for your reply
all the best

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