Coommassie brilliant blue r 250

The substrate gel zymography was performed as previously described [41 (link)] with some modifications. The SDS polyacrylamide gels co-polymerized with gelatin (BioRad Cat. # 170–6537, U.S.A.) (1 mg/ml) were used to detect the gelatinolytic activity in the tissue extracts of knee joints. A total of 26 μg of protein from each sample was subjected to electrophoresis at a constant voltage of 100V. After electrophoresis, gels were incubated in 2.5% Triton X-100 (4 x 15 min changes) with gentle shaking and then incubated overnight at 37°C in activation buffer (50mM Tris-HCl, pH 7.4, containing 10mM Calcium chloride and 0.05% Brij 35 (Sigma, U.S.A.). Gels were then fixed and stained with Coommassie Brilliant Blue R 250 (Sigma, U.S.A.) prepared in fixative (methanol: glacial acetic acid: water:: 45:10:45) and images were acquired on Gel Doc XR+ (BioRad, U.S.A.). The pixel density / unit volume for each band of different active MMPs and ratio between pro / active MMPs for different experimental groups were calculated by using Image Lab software (BioRad, U.S.A.). The identical gels were incubated in activation buffer containing 20mM EDTA (Sigma, U.S.A.), for MMPs inhibition study.

Electrophoresis was performed using the general principle [38 ] and Omni Page electroblotter (Cleaver Scientific). OTf PC2 of the test samples was diluted to a final concentration of 2 mg/mL protein, using 2-mercaptoethanol Laemmli buffer (Sigma Aldrich) and bromophenol blue (Sigma Aldrich). After incubating the samples for 10 minutes at 96°C, 5 μL of each sample was loaded on a polyacrylamide migration gel of 10% and a concentration gel of 4%. A molecular marker, Protein Marker VI (AppliChem), containing a mixture of 12 proteins with molecular weight ranging from 10 to 245 kDa, was also run on the gel. Electrophoresis was performed at 90 mV and 185 mA, for 90 minutes, and staining was performed with Coommassie Brilliant Blue R250 (Sigma Aldrich).

An overnight culture of the recombinant strain ER2523-pMAL-c5X-ipaJ was inoculated into fresh LB medium with ampicillin at a 1:100 dilution. When the OD600 reached 0.4–0.6, IPTG was added to the medium with at a final concentration of 0.3 mM to induce protein expression, and the bacteria cultured at 37 °C for 4 h at a shaking speed of 180 rpm. The bacterial pellets were collected for ultrasonic lysis, the supernatants of lysed bacterial cultures containing the MBP-IpaJ protein were subjected to SDS-PAGE and stained with 0.025% coommassie brilliant blue R-250 (Sigma, USA). Purification of the protein was performed following the manufacture’s instruction by using the pMAL™ Protein Fusion and Purification System (NEB, USA).