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).

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