Anti-Phosphotyrosine Western

1. After SDS-PAGE, set up transfer as usual.
2. Block for 1hr at RT before probing:

   Blocking solution:

   	For 100 ml
   20mM Tris pH 7.4	2 ml 1M Tris pH 7.4
   150mM NaCl	3 ml 5M NaCl
   10% milk	10 g skim milk powder
   1% BSA	1 g BSA
   0.1% Tween 20	100 µl Tween-20
   1mM vanadate	1 ml 100mM vanadate

   ddH20 to 100 ml

3. The remaining steps are performed at RT in 5% milk, 20mM Tris pH 7.4, 150mM NaCl, 1mM vanadate, 0.1% Tween (no BSA).

   Stock solution	For 500ml	For 1 L
   1M Tris pH 7.4	10 ml	20 ml
   5M NaCl	15 ml	30 ml
   skim milk powder	25 g	50 g
   Tween 20	0.5 ml	1 ml
   sodium orthovanadate	0.09 g	0.18 g
   ddH20 to	500 ml	1 L

4. Add antiphosphotyrosine antibody (UBI: 4G10) At 1/5000 (2µL/10ml) For 2 hr.
5. Wash 6x 15 min.
6. Incubate with anti-mouse-HRP conjugated secondary antibody for 1 hr.
7. Wash 5x 10 min with 20mm Tris pH 7.4, 150mm NaCl, 0.1% Tween no milk).
8. Blot membrane On 3mm paper.
9. Place membrane in ECL mix for 1 min. Blot dry on 3mm paper.
10. expose to film for 5 min, develop. Re-expose for various times as necessary.