Westerns without blocking step?
butler at soma.UMDNJ.EDU
Tue Dec 6 08:44:29 EST 1994
T. S. Pillay <tpillay at ucsd.edu> writes:
>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
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
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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