Open AccessDetection of proteolytic activity by covalent tethering of fluorogenic substrates in zymogram gels
Author(s) -
Ameya Deshmukh,
Jessica Weist,
Jennifer L. Leight
Publication year - 2018
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/btn-2018-0005
Subject(s) - proteases , covalent bond , zymography , protease , chemistry , biochemistry , gelatin , collagenase , peptide , electrophoresis , microbiology and biotechnology , matrix metalloproteinase , chromatography , enzyme , biology , organic chemistry
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.