Detection of proteolytic activity by covalent tethering of fluorogenic substrates in zymogram gels

Author:

Deshmukh Ameya A12,Weist Jessica L2,Leight Jennifer L12

Affiliation:

1. Department of Biomedical Engineering, College of Engineering, Ohio State University, 460 W 12th Ave, 504 Biomedical Research Tower, Columbus, OH 43210, USA

2. Comprehensive Cancer Center, James Cancer Hospital & Solove Research Center, Ohio State University, Columbus, OH, USA

Abstract

Current zymographic techniques detect only a subset of known proteases due to the limited number of native proteins that have been optimized for incorporation into polyacrylamide gels. To address this limitation, we have developed a technique to covalently incorporate fluorescently labeled, protease-sensitive peptides using an azido-PEG3-maleimide crosslinker. Peptides incorporated into gels enabled measurement of MMP-2, -9, -14, and bacterial collagenase. Sensitivity analysis demonstrated that use of peptide functionalized gels could surpass detection limits of current techniques. Finally, electrophoresis of conditioned media from cultured cells resulted in the appearance of several proteolytic bands, some of which were undetectable by gelatin zymography. Taken together, these results demonstrate that covalent incorporation of fluorescent substrates can greatly expand the library of detectable proteases using zymographic techniques.

Publisher

Future Science Ltd

Subject

General Biochemistry, Genetics and Molecular Biology,Biotechnology

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