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

1. 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.
2. 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 °C for 1.5 hours or until the molecular weight standards indicate that the proteases of interest are within the peptide resolving gel layer (which has a visible orange color).
   NOTE: Most MMPs and their variants fall within the range of 35–100 kDa. When the molecular weight standards indicate that those weights are within the peptide resolving gel layer, electrophoresis can be stopped. The same principle can be applied to other classes of proteases with known molecular weights. If there is an interest in detecting multiple proteases over a larger range of molecular weights, reduce the size of the resolving gel layer and increase the size of the peptide resolving gel layer.
3. Following electrophoresis, remove the gels from the plastic cassette and wash gels three times for 10 min each at room temperature under gentle agitation in renaturing buffer containing 2.5% Triton X-100, 1 µM ZnCl₂, and 5 mM CaCl₂ in 50 mM Tris-HCl, pH 7.5.
4. Transfer gels to a developing buffer solution containing 1% Triton X-100, 1 µM ZnCl₂ and 5 mM CaCl₂ in 50 mM Tris-HCl, pH 7.5 for 15 min. Replace with fresh developing buffer solution and incubate gels at 37 °C under gentle agitation for 24 hours, making sure the gels are fully submersed in the solution.
5. After 24 hours, image gels using a fluorescent gel scanner/imager using the appropriate excitation and emission filters.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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