Westerns without blocking step?

Gary Butler butler at soma.UMDNJ.EDU
Tue Dec 6 08:44:29 EST 1994


T. S. Pillay <tpillay at ucsd.edu> writes:

>Dear  Netters

>Has anyone tried doing Westerns using Immobilon where the blocking step
>was omitted.  Millipore has been sending fliers around describing this -
>Has anyone tried this?  Does it work as well as the regular extended
>protocols?

I can't answer your specific question as I have not seen the prod. lit.  
However the following may be usefull to you.  If they incorporate 
tween-20 into their antibody buffers and wash buffers as most wb 
procedures do, the tween-20 by itself can function as a background 
blocker.  This may have been discussed in an (5-10yr) old J. Immunol. 
Methods review article (sorry au escapes me).  I tried it once as in a 
straight pre-antibody blocking step., and the signal to noise ratio was 
very good, but the signal was weaker some other blockers.  I had the same 
problem with NFDM (blotto)+ tween, gelatin + tween etc.  The best 
blocking agent for excellent signal and excellent signal to noise ratio 
for me is 10% FBS, 0.05% w/v tween-20, 100 U/ml penn G.  Block for a minimum 
or 3 hours with shaking at RT, and store in refer for use.  Some people
actually use spent FBS-containing culture medium (from lines which don't
make Ig). Never use BSA or BSA with tween.  Pure BSA mediates false 
positives (The stickiness of the BSA in FBS is blocked by the other 
components). I've noticed no great differences among membranes except 
PVDF and Nylon are more durable.  I've also found 1.5 hours to be the 
best time for antibody incubations.  Give the FBS-tween a try if you're 
not using it already - you'll get unequivocal results the fastest.  Good 
luck.  GB
-- 
Gary H. Butler, Ph.D.
Coriell Institute for Medical Research
401 Haddon Ave.
Camden, NJ 08103

Tel: (609) 757-9716
Fax: (609) 964 0254 Attn: Butler
Email: butler at UMDNJ.edu





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