Detection of Cy5 -Reply
mohedley@spiff.pmh.toronto.on.ca
Fri, 03 Nov 1995 07:37:33 -0500
We have quite a bit of experience conjugating antibodies with Cy-5
(obtained from BDS before they were taken over by Amersham) and
measurement using flow cytometry. We found the conjugation to be
generally straightforward. The kit is packaged for 1mg of antibody. Alan
Waggoner recomended reducing the amount of Cy-5 if you have a smaller
amount of antibody - otherwise you can overload the protein and lose
antibody specificity. Unbound Cy-5 is separated on Sephadex G25, so I
doubt if sample line contamination would be a problem. Check dye:protein
by measuring absorbance at 280nm and 650nm (the package tells you
how to do this).
We run with a red HeNe, collecting at 675nm. It doesn't excite at 488nm,
so compensation is no problem, but you need two lasers. The related dye
Cy-3 has spectra similar to phycoerythrin, so does excite with an argon
laser.
David Hedley
Princess Margaret Hospital
Toronto
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