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SDS-PAGE after ammonium sulfate precipitation


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#1 kwesi

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Posted 28 January 2010 - 11:06 PM

I want to check by western blotting if my protein is precipitated with several ammonium sulfate concentrations (35, 50, 60%).

Is it absolutely required to dialyse the dissolved ppt before loaading to a PAGE gel?

Will AS affect the electrophoresis so much? I just want to know if it is precipitated. I will not run the sup, just the ppt

Your responses will be very much appreciated

#2 K.B.

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Posted 29 January 2010 - 03:27 AM

In my experience SDS-PAGE from ammonium sulphate precipitated protein should work and results may be acceptable.

#3 mdfenko

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Posted 29 January 2010 - 07:15 AM

there will be too much residual salt in the pellet. you should dialyze your sample (drop dialysis should be okay).

you could tca precipitate the protein after resuspending the pellet instead of dialyzing but that carries its own post-precipitation manipulations.
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#4 kwesi

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Posted 31 January 2010 - 08:01 PM

thanks for your replies. I will try your suggestions and other options




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