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Measurement of proteasome inhibition in live cells in Molecular Devices microplate fluorometers

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BD Biosciences Clontech has recently introduced the BD Living ColorsHEK 293 ZsGreen Proteasome Sensor Cell Line that allows noninvasive monitoring of proteasome activity.4 It was obtained by stably transfecting HEK 293 cells with the Proteasome Sensor Vector (pZsProSensor1). The Protein Sensor Vector encodes a destabilized version of a green fluorescent protein, (ZsGreen; zFP506.1) that is degraded by the proteasome without the requirement for ubiquitin modification.5 To convert ZsGreen into a proteasomal substrate, its C-terminus was fused to a specific degradation motif that targets the fusion for removal by the proteasome.

Under normal conditions, the fluorescence of the cells is very low because ZsProSensor-1 protein is rapidly degraded. However, under conditions where proteasome activity is inhibited, fluorescence increases as the ZsProSensor-1 protein accumulates. This live cell assay is highly sensitive and can be monitored by fluorescent microscopy, flow cytometry, and microplate fluorometry. Below, we show that inhibition of proteasome activity can easily be measured in the following Molecular Devices microplate fluorometers: The Gemini EM is a benchtop scanning microplate fluorometer with top- and bottom-read capability. The FlexStation is similar, but with integrated fluid transfer capability. The SpectraMax M5 is a scanning multi-detection benchtop reader, also with top- and bottom-read capability. The Analyst GT is a multimode reader designed for high-throughput screening environments.


MATERIALS
HEK 293 ZsGreen Proteasome Sensor Cell Line (BD Biosciences Clontech, Cat. #631535)

Acetyl-leu-leu-norleu-al (ALLN, CalBiochem Cat. #208719)

Black-Wall Clear-Bottom 96-Well Microplate (Costar Cat. #3603)

Gemini EM Scanning Microplate Fluorometer (Molecular Devi
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