Is milk protein Y-phosphorylated?

[dLee]

I routinely use 2% blotto (2% powdered milk in TBS (no tween)) as both
blocking buffer and diluent for anti-PY mAb, and have very clean
backgrounds with very strong signals.  However, I'm using the PY20
anti-phosphotyrosine mAb from Zymed (also available elsewhere) and an
alk phos secondary Ab.

Here is my basic protocol:

1)  Use 5x10^5 cells per well for 10% minigel.

2)  After stimulation, wash cells two times with 10mM Na3VO4.  Lyse
cells in 25ul Triton X-100 with all the typical protease and
phosphatase inhibitors, and typical SDS-PAGE and transfer.

3)  Block minimum 6 hrs, preferably overnight, at 4 degrees.

4) Probe with PY20 (1:1000 in 2% blotto) overnight.

5)  Wash 3x by soaking blot in 10ml TBS-tween for 10 min each, and
rinsing in between washes.  Transfer blot to clean tray.

6)  Probe with secondary Ab, 1:1000 in 2% blotto (AP-GAMIG, Zymed
62-6522) 1hr.

7)  Wash as in 5.

8)  Incubate in BCIP-NBT 10-60 min.

Hope this helps,

Dean Lee
Dept of Micro and Mol Genetics
LLU
dLee at ccmail.llu.edu


P.S.  I sincerely doubt I'll have any trouble publishing these blots.