Judith, Generally normal Ig is used to block Fc receptor binding in your negative control, it is used generally with a nonspecific protein block like BSA and FBS. Isotype control is used as a more specific block to obviate any binding that may be the result of isotype specific binding indeed the best control is an isotype that has the overt specificities of what your attempting to stain and therefore control for, e.g. an anti Ia of k of isotype IgG for an IgG antibody specific for Ia of b. Gene Manager, Flow Cytometry Facility UCONN Health Farmington, CT. 06032 http://flowcytometry.uchc.edu 860 679 7567 -----Original Message----- From: Stewart, Judith [mailto:Judith_Stewart@URMC.Rochester.edu] Sent: Friday, August 12, 2005 8:56 AM To: cyto-inbox Subject: is isotype necessary? I'v been reading the queries lately dealing with titrating, etc. If you properly titrate your antibody to saturation and not beyond, doesn't that mean that there will be minimal binding at any other sites? It would seem that the Keq should give preference to the specific binding site. Isn't that the whole point of titration? So why is an isotype necessary under these circumstances, let alone subtracting the isotype? Am I missing something here?Received on Mon Aug 15 13:58:00 2005
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