Clontech, Takara
Proteasome Sensor Vector (ZsProSensor-1)
632425
We wanted to test proteasome function. So we used Proteasome Sensor Vector ZsProSensor-1 (Clontech #632425). ZsProSensor-1 is a proteasome function sensitive fluorescent reporter that has a bright green fluorescent protein fused with degradation domain. When the proteasome functions normally one can hardly see any accumulation of green fluorescent protein, but if the proteasome is non-functional (like with treatment of proteasome inhibitor MG132) there will be an accumulation of green fluorescent protein. We conclude that Proteasome Sensor Vector ZsProSensor-1 can be used for measurement of proteasome function.
Proteasome function assay
HeLa cells grown on 10 mm glass coverslip.
1. The cells are seeded to about 60% confluency on a glass coverslip. 2. 12 hour later cells are transfected with 0.5 ug of Proteasome Sensor Vector ZsProSensor-1 for 24 hour. 3. Cells were treated with proteasome inhibitor MG132 (20ug/ml) for 6 hour. 4. Cells are washed 3 times with PBS 1x, fixed using 4% paraformaldehyde for 15 mins, washed 3 times with PBS 1x. 5. Coverslips with cells are incubated with Hoechst 33342 10ug/ml for 10 mins and washed with PBS 1x and mounted (using MOWIOL Mounting Media) on to glass slide. 6. Images where acquired on Zeiss 700 confocal microscope
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In the control cells where proteasome inhibitor is not added proteasome functions normally and there is no accumulation green fluorescent protein. But when treated with proteasome inhibitor MG132, proteasome will be non-functional and this led to accumulation of green fluorescent protein inside the cells.
Reagent works well for the assay of proteasome function.
A good reagent.