Reverse phase chromatography of acetone protein precipitate - (Jul/02/2008 )

Dear all,
I would have a question to protein sample preparation for reverse phase chromatography (RPC) protein purification. I want to do RPC protein purification with sample containing my protein (the sample is/are a few fractions from ion exchange chromatography (IEC). I know, that my protein is possible to precipite by acetone from those IEC fractions. So I would do protein acetone precipitation, then I would dissolve the precipitate in guanidine hydrochloride (GnHCl) or urea and I would use it for RPC.
Do you have any experience with the use of protein acetone precipitate (dissolved in GnHCl or urea) for RPC? Does it work well - is the protein separation - resolution O.K. (i.e., comparable with normal acetone non precipited sample)?

Thanks.
vic

-victor.m-

I would try first dissolving in water, if not - in mobile phase A, then in mobile phase B.

-K.B.-

QUOTE (victor.m @ Jul 2 2008, 09:18 PM)

You can dilute the IEC fractions to reduce the salt concentration.

The protein is going to be denatured anyway, so you don't need to worry too much. Just resuspend in Buffer A. Spin down to get rid of any particulate matter and run. If your protein is in the insoluble pellet, add Buffer B until it is soluble, then dilute with A until the concentration of B is low enough to allow binding to the column.

If you're not sure of what will happen re precipitating vs not precipitating the sample, do the experiment.

-swanny-