Fluorescent Peptide Zymography: A Modified Technique to Detect Protease Activity Using Fluorogenic Substrates in Zymogram Gels
Published: April 30, 2023
Abstract
Source: Deshmukh, A. A. et al. Detection of Protease Activity by Fluorescent Peptide Zymography. J. Vis. Exp. (2019)
In this video, we perform zymography, an electrophoretic technique, to analyze the proteolytic activity within biological samples.
Protocol
1. Electrophoresis of Biological Samples in Peptide Zymography Gels Dissolve samples in conventional zymography sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 4% SDS, 0.01% bromophenol blue). For cell and tissue samples, ~30 µg of total protein per well is recommended, and 50–100 ng of protein for MMP standards. Add 400 mL of 1x Tris-Glycine SDS Running Buffer to the gel apparatus. Load up to 35 µL of sample per well. Run the samples at 120 V at 4 °…
Divulgaciones
The authors have nothing to disclose.
Materials
| 1.5 mm Empty Gel Cassettes | ThermoFisher Scientific | NC2015 | |
| 20% SDS Solution | Ambion | AM9820 | |
| 3x Zymography Sample Buffer | Bio-Rad | 1610764 | |
| Azido-PEG3-Maleimide Kit | Click Chemistry Tools | AZ107 | |
| PowerPac Basic Power Supply | Bio-Rad | 1645050 | |
| Precision Plus Protein Dual Color Standard | Bio-Rad | 161-0374 | |
| PrecisionGlide Hypodermic Needles | Fisher Scientific | 14-826 |
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Fluorescent Peptide Zymography: A Modified Technique to Detect Protease Activity Using Fluorogenic Substrates in Zymogram Gels. J. Vis. Exp. (Pending Publication), e20683, doi: (2023).