Kathy, My educated guess would be that it is unlikely that the lack of a signal is due to the fact that EPO receptors are below the level of sensitivity of the instrument which on a FACSan is something of the order of 2000 molecules of FITC if memory serves. More likely I'd think is that your antibody, designed to detect soluble receptor, is specific for an epitope which is only available when the antigen is in soluble form and is hidden when the receptor is bound to the cell surface. I would think you may require an antibody specific for the bound form of the receptor. Gene/UCONN Health ---------- From: Prolifaron@AOL.COM To: cyto-inbox Subject: EPO and receptor numbers Date: Thursday, January 21, 1999 1:14PM I am new to flow and have some basic questions and some specific questions. I would like to examine antibodies to the Epo receptor by flow. I purchased a goat antibody that recognizes soluble receptor in ELISA and western. When I try to examine it on cell surface on TF-1 cells, a human erythroleukemia cell line, I do not detect a signal with either FITC or PE conj secondary antibodies. Can anyone tell me the lower limit for detection of a cell surface protein by FACS? How many receptors per cell do I need to see binding? Also, has anyone out there ever examined EPO receptor by FACS? Can you help me? Thanks, Kathy Bowdish Prolifaron, LLC
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