(1) Rinse blot 3X with WBB (Western Blot Buffer) : 5% dry milk powder, 100 mM NaCl  10 mM HEPES, pH7.5, 0.01% TX-100. Make fresh buffer without azide.

(2) Add primary antibodies at final concentrations of 0.2-1 microgram per mL in WBB.  Incubate a minimum of 2 hours to overnight at at room temperature. 

(3) Wash blot 4 times 15 min each in WBB. 

(4) Add secondary antibodies (horse radish peroxidase attached anti-mouse, anti-rabbit, or anti-goat) at a final concentration of 1 microgram per mL in WBB. Incubate 2 hours at room temperature. 

(5) Wash filters 4 times 15 min each in WBB. 

(6) Wash filters 2 times 10 min each in 100 mM TRIS pH8.5.

(7)  Develop with comaric acid, luminol, peroxide in 100 mM TRIS pH8.5.