Western Blot problems
wgschech at med.uni-tuebingen.de
Thu Aug 20 18:36:05 EST 1998
Actually SDS should be enough to solubilize your G?-protein, at
least when you homegenize your worm in Laemmli as a crude
Normally, 0.5 to 1% triton or nonidet-P40 in 50mM tris pH 7.5
(10mM EDTA, 10mM DTT or mercapto, 150 mM NaCl, lots of protease
inhibitor coctail (Boehringer or Sigma, e.g.) is enough to
solubilize membranes and cytoplasm.
Maybe your AB has problems detecting it. Did you try ECL? You'll find
some good recipes (search for Lumimol and p-Iodophenol) in the NG's
Is it possible for you to include a positive control i.e.
recombinant protein or a known source of good yield?
Make sure that you don't blot too long, some membranes are said to be
critical in respect to blotting times for small molecules. You
might a place two membrane stack in the apparatus.
If you can't load enough material on your gel, try
immunoprecipitation from a lot of solubilized worms.
If you want to discuss more details, feel free to mail me!
usual disclaimers apply * This message is RNAse free - please don't touch!
University of Tuebingen, Germany
email: wgschech at med.uni-tuebingen.de * wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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