At 15:35 22/05/00 -0400, you wrote: > >Hi all >I need to measure EPO receptors in human erythropoietic cell cultures, and >cannot find fluorochrome-conjugated primary antibodies. Does anyone know a >source ? Or is this better done with secondary antibodies ? Many thanks for >any tip. >Ralph Bohmer, New England Medical Center, Boston > > Greetings Ralph, A different approach to this would be to biotinylate EPO and measure cognate receptor expression with a streptavidin/fluorochrome second layer. Pierce market a biotinylation kit for this purpose; the whole procedure takes a few hours as I recall. Having said that, I have to confess I have not tried to biotinylate EPO, I know that G-CSF biotinylates with no apparent loss of activity and I have used it to assay G-CSF receptor expression. The data I have suggests that <100 receptors are detectable by this method. If you are using a murine cell line in this system, you may get a high background staining level due to direct cell/streptavidin binding; you can reduce this by incubating the stained cells with an excess of unconjugated streptavidin or use a modified avidin conjugate 'neutralite avidin' I have used the latter with success on baf3 cells. If 'non-specific' (I know, I know) binding is not a problem, I would recomend 'Streptavidin-RED670' (GibcoBRL cat no. 19543-016) I find it works superbly (2ml gives you about 1000 tests) Regards Arnold _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/ Arnold Richard Pizzey Department of Haematology Royal Free and University College London Medical School 98 Chenies Mews London WC1E 6HX U.K voice: +44 020-7679-6234 Fax: +44 020-7679-6222 email: a.pizzey@ucl.ac.uk _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
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