Westerns without blocking step?

H.J. Treumann Biochemistry ext 4301 hjtreuma at dux.dundee.ac.uk
Wed Dec 7 06:03:14 EST 1994


Dear Gary, 

what is FBS?

Achim Treumann
a.treumann at dundee.ac.uk

Gary Butler (butler at soma.UMDNJ.EDU) wrote:

[... some good advice omitted ...]

: 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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