Western Blot DETECTION OF JUNCTIONAL PROTEINS:
(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
(6) Wash filters 2 times 10 min each in 100 mM TRIS pH8.5.
Develop with comaric acid, luminol, peroxide in 100 mM TRIS pH8.5.