Westerns without blocking step?
H.J. Treumann Biochemistry ext 4301
hjtreuma at dux.dundee.ac.uk
Wed Dec 7 06:03:14 EST 1994
what is FBS?
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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