Detection of Protease Activity by Fluorescent Peptide Zymography

Summary

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

Abstract

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Introduction

Gel zymography is a biological technique used to measure proteolytic activity within biological samples, such as body fluids or cell culture media^1,2,3. The samples are separated by their molecular weights with electrophoresis through a polyacrylamide gel embedded with a degradable substrate. Common degradable substrates include gelatin, casein, collagen and elastin, which have been used to measure the activity of matrix metalloproteinases (MMPs) -1, -2, -3, -7, -8, -9, and -11, in addition to a variety of cathepsins^1,2

Protocol

1. Preparation of the Resolving Gel Layer

1. Prepare a 10% polyacrylamide resolving gel solution as per Table 1. Add the Tetramethylethylenediamine (TEMED) and Ammonium Persulfate (APS) immediately prior to pouring the gel as their addition initiates the polymerization reaction.
2. Fill an empty 1.5 mm mini-gel cassette half way (5 mL) with the 10% resolving gel solution.
3. Add a thin layer of isopropanol (~500 µL) to the top of the polyacrylamide gel to produce a level gel a.......

Discussion

Current zymographic techniques rely on the incorporation of native substrates into polyacrylamide gels for the detection of proteolysis. While these techniques have garnered widespread use, they are still limited in the number of proteases they can detect. Here, a protocol was described in which fluorescent, protease-degradable peptides are incorporated into the polyacrylamide resolving gel. Covalent coupling using an azido-PEG3-maleimide linker molecule enables the separation and detection of a wider variety of protease.......

Materials

Name	Company	Catalog Number	Comments
1.5 mm Empty Gel Cassettes	ThermoFisher Scientific	NC2015
1.5 mm, 10 well Empty Gel Cassette Combs	ThermoFisher Scientific	NC3510
1x Phosphate Buffered Saline	Fisher Scientific	10-010-049
20% SDS Solution	Ambion	AM9820
3x Zymography Sample Buffer	Bio-Rad	1610764
40% (w/v) Acrylamide/Bis (19:1)	Ambion	AM9022
6 Well Tissue Culture Plates	ThermoFisher Scientific	087721B
Amicon Ultra-2 Centrifugal Filter Unit (10 kDa MWCO)	Sigma-Aldrich	UFC201024
Ammounium Persulfate	Sigma-Aldrich	A3678
Azido-PEG3-Maleimide Kit	Click Chemistry Tools	AZ107
Calcium Chloride	ThermoFisher Scientific	BP510100
Dimethyl Sulfoxide	Fisher Scientific	BP231
Isopropanol	Fisher Scientific	A416P
Micro BCA Protein Assay Kit	ThermoFisher Scientific	23235
N N N' N'-Tetramethylethylenediamine (TEMED)	Sigma-Aldrich	T9281
PowerPac Basic Power Supply	Bio-Rad	1645050
Precision Plus Protein Dual Color Standard	Bio-Rad	161-0374
PrecisionGlide Hypodermic Needles	Fisher Scientific	14-826
Round Bottom Flask (100 mL)	Fisher Scientific	50-873-144
Septum Rubber Stopper	Fisher Scientific	50-872-546
Sterile Slip Tip Syringe (1 mL)	Fisher Scientific	14-823-434
Triton X-100	Sigma-Aldrich	X100
Trizma hydrochlroide	Sigma-Aldrich	T5941
Typhoon 9410 Molecular Imager	GE Amersham	8149-30-9410
Zinc Chloride	Sigma-Aldrich	208086