Western Blotting

(Protoblot and Morgan)

General: This protocol is based on Alkaline Phosphatase visualization. It is very sensitive but not very quantative.

Solutions:

TBST pH 8.0 (store at RT) 1 liter (10x)
-10mM Tris-HCl pH 8.0 -10mL 1M
-150mM NaCl -30mL 5M
-0.05% Tween 20 -5mL of 100% stock
Blocking solution
-1% BSA
-4% nonfat dry milk
-1% gelatin
-TBST
3) Alkaline phosphatase (AP) buff (store at RT) 500mL
-100mM Tris-HCl pH 9.5 -6.06g
-100mM NaCl -10mL of 5M stock -5mM MgCl2 2.5mL of 1M stock
4) Western transfer buffer (do not pH) 2liter
-20mM Tris-base -4.82g
-Glycine -32.5g
-20%Methanol -400mL