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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

Development of a high throughput time-resolved fluorescence resonance energy transfer assay for TRAF6 ubiquitin polymerization.

Interleukin 1 receptor activation innervates a cascade of signal transduction events that ultimately lead to the activation of inflammatory and immune response genes. TRAF6 is a Ub ligase ( E3) involved in this pathway, and inhibition of this critical enzyme may provide a means for treating inflammatory and immune diseases. A TR-FRET assay has been developed and evaluated for HTS for TRAF6 inhibitors. Bio-Ub and Eu-Ub were polymerized in the presence of Ub activating enzyme E1, conjugating enzyme E2, and TRAF6. Following a 2-h incubation, the reaction was stopped with a buffer containing 10 m M EDTA and the fluorescence donor SA-APC. Fluorescence energy transfer from Eu to APC was measured as a ratio of fluorescence intensity at 655 nm to that at 615 nm (excitation at 340 nm). This homogeneous assay has been optimized and validated in a 384-well format. A window of five- to eightfold and Z' factor of 0.6-0.8 suggests that this assay can be applied to screen for inhibitors of the polyubiquitination activity of TRAF6.[1]

References

  1. Development of a high throughput time-resolved fluorescence resonance energy transfer assay for TRAF6 ubiquitin polymerization. Hong, C.A., Swearingen, E., Mallari, R., Gao, X., Cao, Z., North, A., Young, S.W., Huang, S.G. Assay and drug development technologies. (2003) [Pubmed]
 
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