| Assay Method Information | |
| | Cell Surface Biotinylation Assay |
| Description: | Huh7.5.1 cells from four 150 mm plates were treated with DMSO, Roc-A (20 nM), or infected by HCVcc. 48 h post-transfection, cells were washed three times with ice-cold PBS and resuspended in PBS at a density of 25×106 cells/ml. Freshly prepared Sulfo-NHS-SS-biotin (Pierce) was added to the cells (final concentration 0.5 μg/ml) and allowed to incubate at 4° C. for 30 min. Cells were then washed three times with ice-cold PBS. 25 mM Tris (pH 8.0) was added in the initial wash to quench any non-reacted biotin reagent. Following cell lysis in RIPA buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 2 mM Na3VO4 and Pierce protease inhibitor cocktail), lysates were cleared by centrifugation at 13,000×g for 15 min at 4° C. The cleared lysates were used for immunoprecipitation using a 1:1 mixture of Streptavidin beads (Pierce). Beads were washed three times with RIPA buffer, and bound proteins were eluted by boiling the samples in SDS-PAGE sample buffer and then resolved on 9% SDS-PAGE. Biotinylated proteins were detected by anti-PHB1 and anti-PHB2 antibodies. |
| Affinity data for this assay | |
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