Binding AssaysThe YHSFFFPGQFAFS peptide derived from chemerin 146-157 was radioiodinated on the tyrosine by the IODOGEN technique and separated from unincorporated radioiodine using a C18 Sep-Pak cartridge. The specific activity of this tracer was estimated to 1250 Ci/mmol. Transfected CHO-K1 cells stably expressing chemerinR were collected from plates with phosphate-buffered saline supplemented with 5 mM EDTA, gently pelleted for 2 min at 1000 x g, and resuspended in binding buffer (50 mM Hepes, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 0.5% bovine serum albumin). A mixture of protease inhibitors (one CompleteTM tablet/50 ml, Roche Diagnostics) was added before use. Binding assays were performed in Minisorb Tubes (Nunc), using 800,000 cells in a final volume of 200µl. For saturation binding assays, increasing concentrations of tracer were used. Total binding was measured in the absence of competitor, and nonspecific binding was measured with an excess (1 µM) of unlabeled ligand. For competition binding assays, 0.5 nM tracer (about 100,000 cpm) was used with variable concentrations of competitors. Samples were incubated for 30 min at 27 °C, then bound tracer was separated by filtration through GF/B filters presoaked in 1% bovine serum albumin. The filters were washed four times with ice-cold binding buffer supplemented with 500 mM NaCl and counted in a scintillation counter. Data resulting from competition binding assays were normalized for the nonspecific binding (0%) and the specific binding in the absence of competitor (100%). Binding parameters were determined with Prism using nonlinear regression applied to a one-site competition binding model. Wittamer et al. J Biol Chem. 2004 Mar 12;279(11):9956-62.
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