Cells were serum-deprived for 4h, then cultured with or without 10μM PE for additional 24h. Media was collected and concentrated at 4°C by ultrafiltration using Amicon Ultra columns (Millipore, Tullagreen, CO). Media equivalent to 30×103 cells was run on 10% gelatin zymography gels (Bio-Rad Laboratories, Hercules, CA). Gels were washed in 2.5% Triton X-100 three times 10min each and incubated overnight in developing buffer (0.05M Tris pH 8.2, 5mM CaCl2, 0.5μM ZnCl2) at 37°C, followed by staining with Coomassie brilliant blue and destaining. Gel images were acquired using Gel Doc LT Imaging Systems (BioImaging Systems; Upland, CA).
Gelatin zymography gels
Manufactured by Bio-Rad
10% gelatin zymography gels are a type of electrophoresis gel used for the detection and analysis of proteolytic enzymes, such as matrix metalloproteinases (MMPs). These gels contain gelatin, which serves as a substrate for the enzymes. After electrophoresis, the gels can be stained to visualize the areas of enzyme activity.
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Lab products found in correlation
2 protocols using gelatin zymography gels
1
Gelatin Zymography Analysis of Matrix Metalloproteinases
2
Gelatin Zymography for Secreted Proteases
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Cells were cultured in serum-free DMEM for 24h. Media was collected and concentrated at 4°C by ultrafiltration using Amicon Ultra columns (Millipore). Media equivalent to 30x103 cells was run on 10% gelatin zymography gels (Bio-Rad Laboratories, Hercules, CA). Gels were washed in 2.5% Triton X-100 three times 10min each and incubated overnight in developing buffer (50 mM Tris pH 8.2, 5 mM CaCl2, 0.5 μM ZnCl2) at 37°C, followed by staining with Coomassie brilliant blue and destaining. Gel images were acquired using Scan Maker i900 (Microtek, Industrial Park Hsinchu, Taiwan).
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