The problem with adding both together is twofold: 1) you may crosslink your biotin-labeled antibody, and potentially cells and 2) you effectively lower the concentration of avidin, since your biotin Ab is in excess, you will bind up some of the avidin with unbound biotin-Ab, which will get washed away. Kevin L. Holmes, Ph.D. Head, Flow Cytometry Unit Office of the Scientific Director Bldg. 7, Room 01 NIAID, NIH Phone: 301-496-9071 FAX: 301-402-4532 Email: kholmes@atlas.niaid.nih.gov -----Original Message----- From: Ioana M Sonea [mailto:ioana@iastate.edu] Sent: Friday, April 23, 1999 2:20 PM To: cyto-inbox Subject: streptavidin question I've been using streptavidin-allophycocyanin to label biotinylated primary or secondary antibodies, in a second step after washing off unbound biotinylated antibody. Has anybody applied streptaviding-conjugated fluorophores at the same time as the biotinylated antibody? It seems to me it should work fine, and it certainly would save time.. Thanks!! ......Ioana Ioana Sonea, DMV, PhD, Adjunct Assistant Professor, Dept. Biomedical Sciences, Iowa State University, Ames, IA 50011 Tel: 515 294 6875 Fax: 515 294 2315 E-mail: ioana@iastate.edu
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