Hi, I need help detecting apoptosis using Apo2.7 antibody. This antibody is specific for a mitochondrial membrane protein that is exposed early in apoptosis. I would like to demonstrate apoptosis on human intraepithelial lymphocytes after activation. But when I use this antibody with the supplied protocol, I get equivalent positivity in all the cell preparations, whether or not apoptosis was induced and regardless of the time point. (Positve compared to an isotype control, that is.) The same thing happens with Jurkat cells: all the cells are positive, even if no anti-Fas is added. I am using the protocol provided by Coulter-Immunotech: -treat cells with 100 mcg/ml freshly-made digitonin in PBS+2%FBS for 20 min on ice, -wash -add .1 ml of PE-apo2.7 diluted 1:5 for 20 min at room temp - Wash twice, keep on ice until analyzed The isotype control is a PE-conjugated mIgG1. Since there is no internal negative, I wonder if I am seeing just background staining that is brighter than that of the isotype control. Does anyone have any experience using this antibody with human lymhocytes? I would appreciate any input. -Art __________________________________ Arthur Roberts Dept. of Medicine Robert Wood Johnson Medical School email: robertar@umdnj.edu phone: 732-235-7790 Fax: 732-235-7792
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