blocking for western blot

Paul S. Brookes. brookes at uab.edu
Fri Apr 21 08:38:27 EST 2000


We rountinely use 10% non-fat dry milk in TBS tween-20, for 1 hour at 37 
degrees.  It's been our experience that blocking overnight at 4 degrees 
requires another 1 hour at room temp' the next morning.  Our "fast" WB 
protocol is as follows...

Block 1 hr, 37C, 10% milk in TBST;  Rinse 2 x 30s. TBST;  Primary Ab, 30 
min., 37C in 5% milk TBST;  Wash 5 x 5 min. TBST; 2nd Ab 30min., 37C in 5% 
milk TBST, Wash 5 x 5 min. TBST, Do ECL.

The milk we use is "Carnation" brand from Wal-Mart (no affiliation ;-p 
).   I think one reason this gives good results is that the time from 
loading the gel to getting the final answer is about 8 hours, so everything 
is still "fresh".  Even a night in the cold room probably doesn't do much 
good for your samples.   BSA works well in our hands too (Sigma fraction V) 
but varies between lots, so buy a big lot and stick with it.

Regards
PSB


_________________________________________
Dr. Paul S. Brookes.            (brookes at uab.edu)
UAB Department of Pathology,   G004 Volker Hall
1670 University Blvd., Birmingham AL 35294 USA
Tel (001) 205 934 1915     Fax (001) 205 934 1775
http://peir.path.uab.edu/brookes

The quality of e-mails can go down as well as up
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