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How to concentrate the protein - (Jul/05/2010 )

hiiiiiiiiii
I need huge concentration of my protein for my assays. For this I lysed transiently transfected 293T cells with 1%SDS and the lysate was further diluted with RIPA buffer containing 1X PBS. the lysate was lyophilized. lyophilized product was dissolved in RIPA buffer. But this protocol is creating problem, becoz of high salt concentration SDS PAGE gel run is not proper(there is a drag,which brings smear). there is not much difference in protein concentration of input and lyophilized sample.

can someone suggest how to concentrate as well as get rid of salts from the lysate. Bcoz at the end of the day I need to send the samples for mass spec.

-sssss-

dint u do dialysis n ion exchange chromatography?

-sunil kumar-

last time I added aceton 50/50 to my total lysate, then rotated overnight at 4C. later centrifuged at 10000 RPM for 15 min. the white pellet was dissolved in lower amount of RIPA compared to first time.

-Curtis-

thanks for your suggestions, actually I am planning to get dialysis kit from pierce, but it will take sometime to get. So, I wanted to know any manual way to concentrate my protein

-sssss-

I used microconcentrators in the past (Amicon, now owned by Millipore).; they worked well:

http://www.millipore.com/catalogue/module.do?id=C7719

-epibio-