Mouse anti human vdr antibody

Distal colonic tissue was placed in lysis buffer containing 120 mM NaCl, 0.5% Nonidet P-40, 0.2 mM sodium orthovanadate, 50 mM Tris–HCl pH 8.0, and 1 Protease Inhibitor Cocktail Tablet per 50 ml (Roche Ltd, Basel, Switzerland). Tissue was sonicated for 5 sec on ice using a Fisher model 60 Sonic Dismembranator (Fisher Scientific, Pittsburgh, PA). Samples were centrifuged at 16,300 × g for 10 min at 4°C and the supernatant was saved. 50 ul of extract from each sample (n=5-6) was pooled per group. VDR and β-actin protein levels were detected in pooled lysates (50 μg) by methods described previously (22 (link),24 (link)). VDR was detected with a mouse-anti-human VDR antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, sc-13133) (25 (link)). The primary antibody was detected with the horseradish-peroxidase-conjugated goat-anti-mouse IgG light chain specific secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and bands were visualized by enhanced chemiluminescent detection (Thermo Scientific, Rockford, IL). Protein bands were quantified using ImageJ software (http://rsbweb.nih.gov/ij/).