I have recently modified my series of web pages on antibody conjugation. Included are new protocols for doing Cy5 direct conjugation (thanks to Tom Waldschmidt for prodding me to use Cy5 on our dye laser!). In addition, I also added protocols for making and conjugating Cy5PE--the brightest of all fluorescent reagents. There are some new tips, especially for separating antibodies on columns. Visit this site at "http://cmgm.stanford.edu/~roederer/abcon/"; it is also on the newest Purdue CD-ROM. In addition, there is now a company, ProZyme, which sells kits to perform PE or APC conjugations. With the kit you get everything you need to conjugate 1 mg of your antibody--including columns, buffers, and the activated phycobiliprotein. And, it only takes about 2 hours--about the same amount of time as it takes to do a simple FITC conjugation. We have used these kits in-house with outstanding results--I highly recommend them to any laboratory that is not prepared to isolate, activate, and crosslink their own phycobiliprotein. Visit ProZyme's web site at "http://www.prozyme.com/phycobiliproteins/". The 1 mg kits cost about $300, which makes them a much more economical choice than custom conjugation (or even buying the pre-made conjugates, if you intend to perform more than a hundred stains!). mr
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