For evaluation of gelatinase activity, cells were grown in serum-containing culture medium on 6-well plates with a change to serum-free medium 24 hours prior to treatment with either CXCL11 (100 ng/ml), CXCL12 (100 ng/ml), or both. Cell supernatant was collected after 48 h and concentrated with Amicon Ultra-2 Centrifugal filter units (Merck Millipore, Burlington, MA). Proteins were loaded (20–30 μg per lane) on polyacrylamide gels containing gelatin (1 mg/ml) and separated by electrophoresis under non-reducing conditions. Gels were subsequently incubated in activation buffer (2.5% Triton X-100, 50 mM Tris HCl, 5 mM CaCl2, 1 μM ZnCl2) for 24 h and stained with Coomassie-Blue for 1 h. Following destaining with methanol/acetic acid, gels were analyzed on a Biostep Celvin S Bioluminescence Detector. Recombinant activated matrix metalloproteinase (MMP)-2 (#550502, Biolegend, San Diego, CA) and recombinant activated MMP-9 (ab81550, Abcam, Cambridge, UK) were used as controls.
Ab81550
Manufactured by Abcam
Ab81550 is a primary antibody produced in rabbit. It is designed for the detection of target protein in various applications.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using ab81550
1
Evaluating Gelatinase Activity in Cells
2
Proteolytic Cleavage of bsGPs by MMP-2
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bsGPs (25 μg) was incubated with recombinant human MMP-2 (Abcam, ab81550) in a final volume of 50 μl in enzyme-specific buffers [50 mM tris, 150 mM NaCl, 5 mM CaCl2, and 1 μM ZnCl2 (pH 7.5)] at 37°C for 120 min. The final MMP-2 concentration was 5 μg/ml.
3
Vitronectin Digestion by MMP-2 and MMP-9
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MMP-2 (ab81550) and MMP-9 (ab82955) were from Abcam. Before incubation with the serum sample, pro MMP-9 was activated by incubation with 4-aminophenylmercuric acetate 1 mM (Sigma, St. Louis, MO, USA) overnight at 37 °C. The digestion of vitronectin was carried out by incubation of the substrate at 37 °C for 24 h with MMPs in an enzyme-to-substrate ratio of 1:20 in 50 mMTris-HCl, pH 7.5, containing 0.15 M NaCl, CaCl2 10 mM, 0.05% Brij 35 and 0.02% NaN3.
4
Gelatinolytic Activity Analysis of MMPs in Fibroblasts
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The gelatinolytic activities related to MMP-2 and MMP-9 were visualised by gelatin zymography in the supernatant from three different culture media of fibroblasts derived from each subjects. The molecular weight markers and samples were electrophoresed under nonreducing conditions by SDS-PAGE in 10% polyacrylamide gels copolymerized with 1% gelatin (Biorad). Before loading, the amount of protein in samples was normalized by DNA content determined spectrophotometrically at A260 after RNA extraction with TRI Reagent (Ambion). All samples were diluted 1 : 1with “Zymogram sample buffer” (Biorad).
After electrophoresis, gels were washed vigorously twice for 15 min in 2.5% Triton X-100 to remove SDS, then incubated in 50 mM Tris/HCl, pH 7.5, 5 mM CaCl2, at 37°C overnight. Gels were stained with 0.5% Coomassie blue G250 for 3 hours. After destaining, the MMP activity was detected as clear bands against the blue background.
The same protocol was carried out to reveal an activity likely related to MMP-3 on 12% polyacrylamide gels copolymerized with 1% casein (Biorad).
An additional zymographic analysis was carried out by loading the active human MMP-2 (Abcam, ab81550, active human MMP-2 full length protein) together with our samples and molecular weight standard. This was done in order to confirm that the most relevant active band we observed in gelatin gel is related to MMP-2.
After electrophoresis, gels were washed vigorously twice for 15 min in 2.5% Triton X-100 to remove SDS, then incubated in 50 mM Tris/HCl, pH 7.5, 5 mM CaCl2, at 37°C overnight. Gels were stained with 0.5% Coomassie blue G250 for 3 hours. After destaining, the MMP activity was detected as clear bands against the blue background.
The same protocol was carried out to reveal an activity likely related to MMP-3 on 12% polyacrylamide gels copolymerized with 1% casein (Biorad).
An additional zymographic analysis was carried out by loading the active human MMP-2 (Abcam, ab81550, active human MMP-2 full length protein) together with our samples and molecular weight standard. This was done in order to confirm that the most relevant active band we observed in gelatin gel is related to MMP-2.
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