PHOSPHOTYROSINE BLOT
Use PY20 monoclonal antibody from Transduction labs. (now owned by Beckton Dickenson)- product data sheet provided for this antibody is kept in the antibodies folder (office)- consult this sheet before proceeding with this method.
Run SDS-PAGE as usual,
and then blot to PVDF membrane (N.B. do not use nitrocellulose)
Wet membrane by immersion in
100% methanol for 15 seconds
then
millipore water for 2 minutes
then
transfer buffer for 10 minutes
Transfer Buffer (1litre)
3.03g Trizma-base
14.4g glycine
200ml methanol
store at 4°C
After transfer- DO NOT STAIN WITH PONCEAU REAGENT!
Block- 1 hour incubation with 1%BSA, 0.1% tween, 10mM Tris, 100mM NaCl pH 7.5
Dilute monoclonal PY20 antibody (1:1000) in blocking buffer and incubate for one hour at room temp.
Wash 6x5 minutes with
10mM Tris ph 7.5, 100mM NaCl, 0.1% tween (WB)
2° antibody- incubate with anti-mouse (1:1000) in blocking buffer.
Wash 6x 5 minutes with WB