RE: kinases PY-blot

[Bart Sefton <sefton@salk.edu>]

Dear Marianne

One of your problems is the skim milk.  Mark Kamps found that milk gives
a high background with anti-phosphotyrosine antibodies.  It is better to
use 3% BSA.  The reference for this is:

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=2452398&form=6&db=
m&Dopt=b

Additionally, if you are blotting lysates, you will get a lower
background if you use iodinated protein A as a detection reagent.  ECL tends to
stain the most abundant proteins in a lysate and this can obscure a weak
anti-phosphotyrosine signal.

Good Luck
Bart