Streptavidin-R-PE

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	Use of 1/1000 dilution

Protocol tips
Use of 1/1000 dilution

Publication protocol

Analysis of cellular E2 and HCV-LP binding.
Binding of recombinant soluble E2 to PTH was performed as described recently (35, 36). Briefly, freshly isolated PTH (105 cells per assay) were incubated with E2 for 1 h at room temperature. Cells were washed two times and incubated with a biotinylated anti-penta-His mouse antibody directed against the His tag of recombinant E2 (1:50 dilution) for 1 h at room temperature. The cells were washed again, and cell-bound antibodies were detected by the addition of streptavidin-R-PE (1:100 dilution) binding to biotin residues of the primary antibody. Flow cytometry was performed using FACSCalibur (Becton Dickinson) and analyzed with CellQuest software. For the measurement of HCV-LP binding, freshly isolated PTH (105 cells per assay) were incubated with HCV-LPs or insect cell control preparation (derived from insect cells infected with a recombinant baculovirus containing the cDNA for β-glucuronidase) in PBS for 1 h at 4°C. Cell bound HCV-LPs were analyzed by FACS as described previously using either mouse monoclonal anti-E2 (16A6) and R-PE-conjugated goat anti-mouse IgG antibodies or chimpanzee monoclonal anti-E2 (49F3) and R-PE-conjugated goat anti-human IgG antibodies (41). To study whether cellular binding of recombinant E2 was inhibited by anti-SR-BI antibodies, cells were preincubated with different dilutions of anti-SR-BI for 1 h at room temperature prior to the addition of recombinant E2 and E2 binding was detected using biotinylated anti-penta-His mouse antibody and streptavidin-R-PE (1:100 dilution). Since this assay does not use dye-conjugated secondary antibodies for the detection of E2, it can easily distinguish between bound nonbiotinylated mouse anti-SR-BI (not reacting with streptavidin-PE) and biotinylated anti-His-E2 (specifically interacting with streptavidin-PE). To study whether cellular binding of HCV-LPs was inhibited by anti-SR-BI antibodies, cells were preincubated with different dilutions of mouse anti-SR-BI for 1 h at room temperature prior to the addition of recombinant HCV-LPs, and HCV-LP binding was detected using chimpanzee monoclonal anti-E2 (49F3) and R-PE-conjugated goat anti-human IgG antibodies.

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