From: T. Vincent Shankey (Thomas Shankey) (TSHANKE@wpo.it.luc.edu)
Date: Fri Apr 17 1998 - 19:42:18 EST
Keith, Provided the fluorescein actually was conjugated to the antibody, the off rate should be quite low. However, there is also the possibility that the "conjugate" contains much free (unconjugated) fluorescein. You could test this by running the "conjugate" through a small column of Sephadex G-25 or G-50. A final possibility (not exclusive of the above) is that your target intracellular antigen is present in large amounts- consistent with your flow results. A similar pattern is seen staining intermediate filaments, ie cytokeratins. We have never been able to decrease the mean ch fluorescence by mixing "unlabelled" antibody with FITC anti-cytokeratin (this also assumes the labelled and unlabelled antibodies are equavalent). In addition to the large number of target molecules, the antibody manufacturese (and you know who you are) tend to go for high F:P ratios - good for surface antigens not present in high copy number, but a problem when you want to saturate (ie quantify) a high copy molecule like cytokeratin. Regards, Vince Shankey
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