acetone precipitation of samples (for SDS PAGEs)

Jesus Sanz 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:
>
>>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 --->
>
>        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

Hi,

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.

Jesus




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