Western Blot Detection:		3/98
Detection of biotinylated 2'Ab with Vectastain ABC-AP.

1. Rinse blot in H2O.
2. Block non-specific sites on the membrane with 5%(w/v) milk plus 
	0.1% (v/v) Tween 20 in TBS at 4'C O/N or 1 hour at RT.
3. Dilute 1' Ab 1/100 to 1000 or as appropriate in Blocking Buffer.
4. Incubate 1 hour at room temperature.
5. Wash filter 3X/RT/15 minutes in T-TBS.
6. Dilute biotinylated 2'Ab (ex: goat anti rabbit) as appropriate in Blocking Buffer.
7. Incubate for 1 hour at room temperature.
8. Wash 3X/RT/15 minutes in excess T-TBS.
9. Dilute Vectastain (two drops A then B per 5 ml) in T-TBS and let 	stand for 
	at least 30 minutes before use.
10. Incubate membrane in Vectastanin for 30 minutes at RT.
11. Wash 3X/RT/15 minutes.
12. Dilute Substrate by adding 45 ul of NBT and 35 ul BCIP (X-phos) in
	10 ml of Detection Buffer.
13. Equilibrate membrane in Detection Buffer for 1-2 minutes.
14. Incubate membrane in Substrate for 30 minutes or until blue precipitate forms.

Detection Buffer: 1 M Tris-HCl, pH 9.5, 1 M NaCl.
Tris buffered Saline with Tween 20 (TTBS): 0.1 M Tris HCl, pH 7.5, 0.1 M NaCl plus 0.1% (w/v) Tween 20.
Blocking Buffer: 5% (w/v) Dried powdered milk, 0.1% (v/v) Tween 20.