acetone precipitation of samples (for SDS PAGEs)
espinoza at cgl.ucsf.edu
Mon Nov 18 15:53:57 EST 1996
siyer at bmg.bhs.uab.edu (Sai) writes:
> 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 --->
Different proteins precipitate in different concentrations of
acetone. I would suggest you try a range of acetone concentrations
(20-80%) and go with the minimum that precipitates your protein. No
guarantees, but it could help.
Alternatively, try TCA precipitations. The wisdom that was
passed on to me was :
-Add 1/5th volume 100% TCA
-Incubate on ice 30'
-Spin 5' in a microfuge (14,000k) @ 4C
-Resuspend in a minimal volume of SDS Loading Buffer
which will turn yellow from the TCA.
-Neutralize sample with 2M Tris (un - pH'd) so it
turns blue again
-Load that sucker.
Good Luck. -Hernan
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