Sample prep for IEF-PAGE?

OBF het_obf at rediffmail.com
Fri Jan 23 10:32:54 EST 2004


Hi everybody, 

I am planning to do some experiments to determine the phosphorylation
state of a 15 kDa regulator in my bug (a Bacillus sp.).
I am intending to use IEF-PAGE to study this. The pI of the regulator
should change upon phosphorylation and so I should be able (in
principle) to seperate the two form by IEF-PAGE and then I can detect
the protein by Western blotting. I will be using Bio-Rad's Criterion
system and their precast gels.
My question now is how I should treat the protein samples prior to
loading. Can I just extract total protein extracts (with beadbeating)
and then I plan to resuspend an aliquot of the extract in loading
buffer (50% glycerol) and start the electrophoresis. Or should I first
pretreat my sample to get rid of membrane/DNA/salts etc?
Any advice would be much appreciated. Also links to appropriate
websites/books/articles!

Regards, 
Willem



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