Identification of Specific Relaxin: MATERIALS AND METHODS(3)
The reaction mixture was stirred at room temperature for 4 h, and the reaction was stopped by the addition of acetic acid until a 1 M acetic acid solution was obtained. The contents of the reaction mixture were separated from the biotinyl-S-amino hexanoyl-relaxin (biotinylated relaxin) by ultrafiltration using an Amicon model 402 stirred ultrafiltration apparatus with a Diaflo Ultrafilter type YM1 membrane (molecular weight cut-off 1000; Amicon, Beverly, MA). The N-methylmorpholine-HCl buffer and acetic acid were replaced with PBS (0.01 M NaH2 PO4 and 0.15 M NaCl, pH 7.4) in the ultrafiltration unit. The biotinylated relaxin was stored at a final concentration of 9 nmol/ml at – 70°C. buy cipro
The mean number of biotin molecules per biotinylated relaxin molecule was found to be 3.5 when determined by a spectrophotometric 4′-hydroxyazobenzene-2-carboxylic acid (HABA) assay according to the manufacturer’s protocol (Pierce Chemical Co., Rockford, IL). The bioactivity of biotinylated relaxin was determined with a mouse interpubic ligament bioassay, and it did not differ statistically from that of unmodified porcine relaxin.
Tags: Cells, Female, Human, Relaxin