Western Blot problems

Wolfgang Schechinger wgschech at med.uni-tuebingen.de
Thu Aug 20 18:36:05 EST 1998


Hi Andreas, 

Actually SDS should be enough to solubilize your G?-protein, at 
least when you homegenize your worm in Laemmli as a crude 
start. 
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 
archive. 

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!

Good luck!

Wolfgang
-----
usual disclaimers apply * This message is RNAse free - please don't touch!
-----                              
Wolfgang Schechinger         
University of Tuebingen, Germany
email: wgschech at med.uni-tuebingen.de * wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
-------



More information about the Methods mailing list