Help needed about western

[Michael W. Thompson]

      Nitrocellulose is a different critter than PVDF.  I routinely use nitrocellulose (In my 
opinion I get less background) and after the wet transfer, you MUST allow the membrane 
to air dry at least one hour, otherwise most of the protein will be washed off during the 
blocking step.  After drying the membrane, simply re-wet the membrane in the blocking 
buffer.  It's not necessary to use methanol (in fact, you DON'T want to use methanol after 
the transfer).  After labelling with the secondary antibody, you'll need to wash the 
membrane very extensively in TBST or whatever you're using (I use PBST, personally) 
for at least 30-45 minutes, or you'll get background streaking on your membrane)
     Another problem I have seen with 'disappearing proteins' is that sometimes when you 
shake the membrane too hard during blocking or any of the subsequent washes or antibody 
labellings, loosely-bound protein CAN sometimes come off the membrane.  So gentle 
shaking (or preferably, a rocker platform set on a very slow speed) is preferable.

Hope this helps.

Michael W. Thompson
Dept. of Biochemistry
University of Kentucky
Lexington, KY 40536-0084