Document Detail

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.
MedLine Citation:
PMID:  23532386     Owner:  NLM     Status:  MEDLINE    
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.
Jerald E Dumas; Manu O Platt
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Molecular biotechnology     Volume:  54     ISSN:  1559-0305     ISO Abbreviation:  Mol. Biotechnol.     Publication Date:  2013 Jul 
Date Detail:
Created Date:  2013-05-02     Completed Date:  2013-11-04     Revised Date:  2014-07-02    
Medline Journal Info:
Nlm Unique ID:  9423533     Medline TA:  Mol Biotechnol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1038-47     Citation Subset:  IM    
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MeSH Terms
Cathepsins / analysis*,  isolation & purification
Cellular Phone
Densitometry / instrumentation,  methods*
Electrophoresis, Polyacrylamide Gel / methods*
Histological Techniques / economics,  instrumentation,  methods
Image Processing, Computer-Assisted / methods
Limit of Detection
Lung / chemistry
Neoplasms / enzymology*
Photography / instrumentation
Time Factors
Grant Support
Reg. No./Substance:
EC 3.4.-/Cathepsins

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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