Rabbit anti mouse peroxidase conjugated secondary antibody

Non-denaturing PAGE was modified with the Laemmli method [21] (link). Electrophoresis was implemented using a 10% (w/v) polyacrylamide resolving gel and a 5% (w/v) polyacrylamide stacking gel. After electrophoresis, proteins in the gel were visualized with Coomassie Brilliant Blue. For zymogram analysis, gelatin (0.1%) copolymerized with the acrylamide gels was used. Electrophoresis was performed at 4°C at a constant voltage of 100 V. The gels were incubated in HEPES (50 mM) buffer for 2 h at 4°C and then in Tris-HCl buffer (pH 8.0) for 3 h at 70°C. The gels were finally soaked in 20% ice-cold trichloroacetic acid for 1 h and stained with Coomassie Brilliant Blue. Proteolytic activity was indicated by the formation of clear zones in the gel containing gelatin. For western blot, the proteins were separated by 10% non-denaturing PAGE and transferred onto a polyvinylidene fluoride membrane. The membrane was probed with the His-Tag TM monoclonal antibody (Merck, San Diego, CA) at a dilution of 1∶1000 in tris-buffered saline (TBS) for 2 h, followed by the rabbit anti-mouse peroxidase-conjugated secondary antibody (Merck, San Diego, CA) at a dilution of 1: 5000 in TBS for 1 h. The blot was developed using a 3,3-diaminobenzidine tetrahydrochloride solution.

Restriction enzymes were purchased from Promega (Madison, WI). Pfu DNA polymerase was supplied by Stratagene (Madison, WI). Ampicillin and isopropyl thio-β-D-galactopyranoside (IPTG) were obtained from TaKaRa Shuzo (Otsu, Shiga, Japan). The 7-amino-4-methylcoumarin (amc)-linked substrates L-D-amc, L-S-amc, L-V-amc, L-A-amc, L-F-amc, L-R-amc, and hippuryl-R were purchased from GL Biochem (Shanghai, China). The His-Tag™ monoclonal antibody and the rabbit anti-mouse peroxidase-conjugated secondary antibody were purchased from Merck (San Diego, CA). Other chemicals used were reagent-grade. The pET-15b vector and the E. coli strain were purchased from Novagen (Madison, WI).