Klotho MAb: Hybridoma Lines KL-115 and KL-234

摘要

Human klotho was PCR amplified using a pcDNA3.1 plasmid containing the cDNA across the sequence corresponding to amino acids 55-261. The resulting fragment was cloned into pGEX-6P2 (GE Healthcare, Piscataway, NJ) in frame with glutathione-S-transferase (GST) at the BamHI/XhoI restriction sites. Plasmid was transformed into Escherichia coli strain BL21 (Agilent, San Jose, CA) and GST-klotho was induced by the addition of 0.4 mM IPTG for 16 h at room temperature. Bacteria were pelleted, lysed in STE (100 mM NaCl, 10 mM Tris [pH 7.6], 1 mM EDTA). Lysates were treated with lysozyme (10 mg/mL) for 15 min followed by the addition of 5 mM DTT, and 1.5% Sarcosyl.