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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 media1,2,3. 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 cathepsins1,2

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.......

Representative Results

Using the method described here, two fluorescent protease-degradable peptides were incorporated into polyacrylamide gels: GGPQG↓IWGQK(PEG)2C (abbreviated as QGIW throughout the text and figures) and GPLA↓CpMeOBzlWARK(PEG)2C (abbreviated as LACW throughout the text and figures). ↓ indicates the site of cleavage. QGIW is a collagen-I derived sequence designed to detect cellular collagenases14. LACW is a sequence that.......

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.......

Acknowledgements

Funding provided by The Ohio State University College of Engineering, Biomedical Engineering Department, and the Comprehensive Cancer Center - Arthur G. James Cancer Hospital and Richard J. Solove Research Institute.

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Materials

NameCompanyCatalog NumberComments
1.5 mm Empty Gel CassettesThermoFisher ScientificNC2015
1.5 mm, 10 well Empty Gel Cassette CombsThermoFisher ScientificNC3510
1x Phosphate Buffered SalineFisher Scientific10-010-049
20% SDS SolutionAmbionAM9820
3x Zymography Sample BufferBio-Rad1610764
40% (w/v) Acrylamide/Bis (19:1)AmbionAM9022
6 Well Tissue Culture PlatesThermoFisher Scientific087721B
Amicon Ultra-2 Centrifugal Filter Unit (10 kDa MWCO)Sigma-AldrichUFC201024
Ammounium PersulfateSigma-AldrichA3678
Azido-PEG3-Maleimide KitClick Chemistry ToolsAZ107
Calcium ChlorideThermoFisher ScientificBP510100
Dimethyl SulfoxideFisher ScientificBP231
IsopropanolFisher ScientificA416P
Micro BCA Protein Assay KitThermoFisher Scientific23235
N N N' N'-Tetramethylethylenediamine (TEMED)Sigma-AldrichT9281
PowerPac Basic Power SupplyBio-Rad1645050
Precision Plus Protein Dual Color StandardBio-Rad161-0374
PrecisionGlide Hypodermic NeedlesFisher Scientific14-826
Round Bottom Flask (100 mL)Fisher Scientific50-873-144
Septum Rubber StopperFisher Scientific50-872-546
Sterile Slip Tip Syringe (1 mL)Fisher Scientific14-823-434
Triton X-100Sigma-AldrichX100
Trizma hydrochlroideSigma-AldrichT5941
Typhoon 9410 Molecular ImagerGE Amersham8149-30-9410
Zinc ChlorideSigma-Aldrich208086

References

  1. Vandooren, J., Geurts, N., Martens, E., Vanden Steen, P. E., Opdenakker, G. Zymography methods for visualizing hydrolytic enzymes. Nature Methods. 10 (3), 211-220 (2013).
  2. Toth, M., Fridman, R. Assessment of Gelatinases (MMP-2 and MMP....

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Protease ActivityFluorescent Peptide ZymographyFluorescent PeptidesDegradable SubstrateModular DesignTissue Secreted ProteasesBiomaterial DegradationPolyacrylamide GelAzido PEG3 MaleimideInert Gas

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