Is milk protein Y-phosphorylated?

Jim Woodgett jwoodget at ocicl.oci.utoronto.ca
Thu Jan 20 14:18:18 EST 1994


In article <2hm67g$amu at charm.magnus.acs.ohio-state.edu>, 
gchacko at magnus.acs.ohio-state.edu (George W Chacko) writes:
 
> I observe from a few experiments where I immunoblot with mab 4G10 
> (anti phosphotyrosine) and detect with GAM_HRP_ECL I get a diffuse 
> background that I don't see when I blot with other mouse monoclonals 
> or rabbit polyclonals. If I use 5% non fat dry milk dissolved in tris 
> buffered saline with 0.01% Tween 20 (TBS-T) as both a blocking agent 
> and a carrier for 4G10 I lose some of the signal and almost all of the 
> background. If I blot with the antibody in TBS-T I get improved signal 
> but the nasty looking background returns and is difficult to wash 
> away. 
> 
> 1) Some component of the dry milk is competing away my signal. 
> 2) Is that component phosphorylated tyrosine? 
> 3) Would substituting BSA for milk help? Or is there some way of 
> preblocking or chemically treating the carrier? 
> 
> Does anyone else have thoughts or experiences on the subject? 
> 
> George Chacko 

1. Essentially, yes.
2. Yes.  Casein contains phosphotyrosine.
3. Yes.  Use BSA or ovalbumin.  It's more expensive but works.  As the casein 
is a rather major component of milk, enzymatic dephosphorylation is out and 
alkali treatment is too much trouble (in dealing with the denatured protein 
upon reneutrilizing the solution).

The blocking agents and conditions for anti-pTyr blotting can be quite 
variable depending on the antibody.  If you bought the antibody from a 
company, the optimal conditions have probably been determined by them.  If 
not, check out a review by Jean Wang in Anal. Biochem. 172, 1-7 (1988).  Mark 
Kamps and Bart Sefton also published a really useful paper on the technique 
(I think in Anal. Biochem.) around 1988.

Jim







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