Fluorescent Peptide Zymography: A Modified Technique to Detect Protease Activity Using Fluorogenic Substrates in Zymogram Gels

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 °…

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