Hello--I am about to begin my first ever flow experiment, and I have questions regarding the staining of the protein (PARP [poly(ADP)ribose polymerase]) with the Abs. I have a FITC-conjugated rat anti-mouse IgG1 Ab (secondary Ab) and a separate mouse anti-human PARP Ab (protein specific Ab). The only published protocol utilized a single FITC conjugated anti-PARP Ab. So, how do I proceed with the separate secondary FITC-conjugate Ab and the separate protein specific Ab. Specifically: 1) Should I perform a separate incubation step for the seconday Ab then the PARP specific Ab; or are they incubated together? 2) If incubated together, how long? (original protocol was 45 min at 4 C), 3) Next, how do I determine the proper concentrations/volumes for each Ab (the published protocol stated 20 ul [5 ug] of the FITC-conjugated Ab was used). Any information would be greatly appreciated. Thanks, Matt. ================ Matthew B. Schabath Predoctoral Fellow Department of Epidemiology M.D. Anderson Cancer Center lab: 713-792-5760 office: 713-745-1078 fax: 713-792-0807
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