acetone precipitation of samples (for SDS PAGEs)

David C. Logan david.logan at plant-sciences.oxford.ac.uk
Sun Nov 17 13:50:41 EST 1996


Sai Iyer wrote:

>hey all, 
>i've posted this before, but i figured i'd try again to see if
>there were any other ideas.  i've been doing acetone precipitation to
>remove salts from my samples prior to loading on sds gels.  the basic
>protocol is add 8 vol. of acetone to 1 vol. of protein sample --->
>precipitate at -20 degrees for 4-6 hours ---> spin down for 20 min at
>14,000g (max speed on centrifuge) at 4 degrees to pellet out the
>precipitated protein ----> decant the supernatant and immediately
>solubilize the pellet in sds loading buffer.  the problem is see is 
>that i still get streaks in my gels.  theoretically, acetone is an 
>uncharged organic solvent and so should not create any distortions in 
>the gel cuz
>its not charged.  so if there were any residual acetone left, that 
>still
>shouldnt create the streaks...yet i still see streaks.  anybody have 
>any
>ideas on this?  or other methods to remove salts from samples (i.e tca
>precipitations etc) would also be welcome...thx...cheers....

There should be no problem using this general method - I suggest your 
particular problem is due to the fact that you are not allowing time for 
residual acetone in the protein pellet/bottom of the tube to evaporate. 
Therefore, allow an empirically determined drying time after the 
centrifugation step before resuspension in SDS loading buffer (approx. 
15 mins should do the trick).

David 
***************************
david.logan at plants.ox.ac.uk

David C. Logan
Department of Plant Science
University of Oxford
South Parks Road
Oxford
OX1 3RB

***************************



More information about the Methods mailing list