Dear Flowers, I am attempting to analyze surface and intracellular levels of the TGF-beta type II receptor (RII) by flow and intracellular flow. I am using a biotinylated primary antibody (a goat polyclonal unfortunately) and then coming back with a stretavidin comjugate (APC -strep). For surface staining, I do get a nice shift relative to a "normal" biotinylated goat polyclonal (my "isotype" control). For the intracellular staining, even when I pre-incubate (in permeabilization buffer, containing saponin) with unconjugated streptavidin (to reduce background) I still see a significant shift with my biotinylated "control". The MFI is around the 3rd log decade, which is bright. No further shift is seen with the biotinylated anti-RII. To my knowledge, no directly conjugated primary antibodies are available against the extracellular domain of RII. Does anyone out there have any experience in: (1) looking at surface or IC TGF-beta type II receptor levels by flow cytometry; &/or (2) using biotinylated antibodies in surface staining or IC flow? Any help would be greatly appreciated. Thanks, Lawrence Allen Wolfraim, Ph.D. Laboratory of Cell Regulation & Carcinogenesis (LCRC) National Cancer Institute/National Institutes of Health 41 Library Drive Building 41, Room B910 Bethesda MD 20892 Tel.: (301) 496-2431 Fax: (301) 496-8395 wolfrail@mail.nih.gov <mailto:wolfrail@mail.nih.gov>
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