Western protocol (Protoblot and Morgan)

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

Solutions:

1. 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

1. 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