Molecular Biotechnology

, Volume 54, Issue 3, pp 1038–1047 | Cite as

Systematic Optimization of Multiplex Zymography Protocol to Detect Active Cathepsins K, L, S, and V in Healthy and Diseased Tissue: Compromise Among Limits of Detection, Reduced Time, and Resources

  • Jerald E. Dumas
  • Manu O. PlattEmail author


Cysteine cathepsins are a family of proteases identified in cancer, atherosclerosis, osteoporosis, arthritis, and a number of other diseases. As this number continues to rise, so does the need for low cost, broad use quantitative assays to detect their activity and can be translated to the clinic in the hospital or in low resource settings. Multiplex cathepsin zymography is one such assay that detects subnanomolar levels of active cathepsins K, L, S, and V in cell or tissue preparations observed as clear bands of proteolytic activity after gelatin substrate SDS-PAGE with conditions optimal for cathepsin renaturing and activity. Densitometric analysis of the zymogram provides quantitative information from this low cost assay. After systematic modifications to optimize cathepsin zymography, we describe reduced electrophoresis time from 2 h to 10 min, incubation assay time from overnight to 4 h, and reduced minimal tissue protein necessary while maintaining sensitive detection limits; an evaluation of the pros and cons of each modification is also included. We further describe image acquisition by Smartphone camera, export to Matlab, and densitometric analysis code to quantify and report cathepsin activity, adding portability and replacing large scale, darkbox imaging equipment that could be cost prohibitive in limited resource settings.


Cathepsins Zymography Detection Cysteine protease Cancer 



This study was funded by the Georgia Cancer Coalition (M.O.P.) and the Institutional Research and Academic Career Development Awards (IRACDA Grant Number K12 GM000680, NIH/NIGMS) (J.E.D.).


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Wallace H. Coulter Department of Biomedical EngineeringGeorgia Institute of Technology and Emory UniversityAtlantaUSA

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