Is milk protein Y-phosphorylated?

Curtis L. Ashendel ashendel at aclcb.purdue.edu
Thu Jan 20 14:07:34 EST 1994


On 20 Jan 1994 14:58:56 GMT, George W Chacko wrote:

>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?

This problem with the use of milk (and Tween, by the way) was noted by 
Kamps and Sefton in their original (?) description of anti-pY antiserum 
(Kamps, M. P. amd Sefton, B. M.  Oncogene 2:  305-315, 1988.)
They were unable to determine if milk has pY in it.  It does not matter.  
You will (IMHO) never get a figure published in the reviewed literature if 
it is of an anti-pY blot done with milk as a blocking agent. 

We us BSA (50 mg/ml) in TBS as block and incubation medium.  This results 
in a higher background, which can largely be eliminated by much more 
extensive washing: 1 wash for 60 min followed by 4 more for 10 min each 
with a minimum of 300 ml wash per 11 x 14 cm blot.  Rinsing between washes 
also helps reduce the background.  If the signal is there but the 
background is too high, try reducing the conc of the primary Ab or 
secondary Ab.  We use ECL with proteinA-HRP secondary and have to dilute 
the secondary by 3 to 5-fold more than usual or the backgound is 
intolerable.

I tested many combinations of blocking agents, antibody concentrations, 
and washing methods, and this was the best I could do.  If you want to do 
some optimizing yourself, I recommend that you do it first using a sure-
fire positive control, such as extracts of v-src over-expressing cells, 
then go on to your experimental samples.
Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu



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