acetone precipitation of samples (for SDS PAGEs)
cibsm1s at fresno.csic.es
Wed Nov 20 13:18:44 EST 1996
In article <espinoza.848350437 at cgl.ucsf.edu>, espinoza at cgl.ucsf.edu (Hernan Espinoza) says:
>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
I've used TCA precipitation and worked fine for me using the above
protocol (more or less) but I've found better results washing the
pellet with ethanol:ether (1:1) to remove any traces of TCA and leave
drying well. Then, you do not need to neutralize the sample.
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