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WESTERN HYBRIDIZATION PROTOCOL
There are many kits available for Western hybridization studies, including an indirect alkaline phosphatase kit from Biorad (1706509, 170-6511) and an avidin-biotin-phosphatase linked kit from Vector Laboratories. Both come with very detailed, easy to use instructions. I have found the Biorad kit to be very simple, and adequate for most purposes, and I will not repeat the protocol here. The Biorad kit uses a TBS/Tween buffer system, gelatin to block, an alkaline phosphatase conjugated secondary antibody (goat anti-mouse, goat anti-rabbit;), and a BCIP/NBT developing reagent. The Vector Laboratories kit uses a PBS/serum blocking/buffer system, with a biotinylated secondary antibody, an avidin-biotin phosphatase linked complex, followed by an 'unknown' development reagent (I have used substrate kit II,black, SK-5200). In addition to these hybridization protocols, I have tried two variations of the Biorad protocol, a powdered milk based buffer system and a serum/PBS buffer system. I have found the powdered milk protocol to work about as well as the standard Biorad protocol, although the serum\PBS protocol appears to be superior (at least for the antibodies I have used) to the standard protocol. The serum\PBS modification is a bit easier than the standard Biorad protocol:
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