Anti-phosphotyrosine immunoblot

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Fri Aug 23 02:58:03 EST 1996


John Hines (hines at pharm.med.upenn.edu) wrote:
> Am I the only one with such problems with ECL??

No. Occasionally ECL gives strange blots (i.e. plenty of background). 
This may depend on your blocking conditions (we use 2% milk 1% ovalbumin
and incubate our 1st antibody usually in this solution; you may also
try to increase blocking time, e.g. overnight). If PVDF membranes
are not preactivated properly, they may also yield blots with incredibly
high background. Moreover, you might think of switching your secondary
antibody (isn't PY20 a mouse monoclonal?), and occasionally, you may
get a bad batch of the kit (which, however, in our hands then results
in no bands/background at all). ECL is much more sensitive than
colorimetric detection, so it's not too surprising that you don't get
any bands with the latter.

All IMO, since I have no experience whatsoever with PY20.

--Cornelius.

-- 
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP3 */
/* "Science is the game we play with God to find out what His rules are."  */



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