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JoVE Journal Bioengineering
Quantitative FRET (Förster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination
 

Quantitative FRET (Förster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Article DOI: 10.3791/4430-v • 17:12 min • February 21st, 2013
February 21st, 2013 •
1, 1
1Department of Bioengineering, Bourns College of Engineering, University of California, Riverside

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A novel method involving quantitative analysis of FRET (Förster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

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Quantitative FRET Analysis F&246;rster Resonance Energy Transfer SENP1 Protease Kinetics Determination Posttranslational Modifications Ubiquitin Ubiquitin-like Proteins Protein Activity Regulation SUMO Modification Cellular Processes SUMO-specific Protease Endopeptidase Isopeptidase Catalytic Efficiency Specificity Kinetic Constants Kcat/KM Ratio Methods For Determining Kinetic Parameters Polyacrylamide Gel-based Western-blot Radioactive-labeled Substrate Fluorescent Compound Protein Labeled Substrate
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