Proteasome Assay to Test Caspase-Like Activity
The kit used for this assay is as follows:
Proteasome-Glo Caspase-Like Assay
Promega
Cat # G8641/2
Lot# 229815
This kit contains:
Proteasome-Glo Buffer (10ml) (G854A)
Luciferin Detection Reagent (1 Bottle) (V859A)
Z-nLPnLD-Glo Substrate (50µl) (G857A)
The day before the assay:
-
plate HT-22 and CHIP OE cells in 6-well plates at a density of 100,000
cells per well
incubate at 37ºC over night
The day of the assay:
-
Resuspend Luciferin Detection Reagent in 10ml of the Proteasome-Glo
Buffer
Prepare Proteasome-Glo caspase-like assay reagent by adding 50µl of ZnLPnLD-Glo susbstrate into the luciferin detection reagent
Incubate the proteasome-glo-caspase-like assay reagent at RT for 60min
to remove unconjugated aminoluciferin
Into a white 96-well plate add equal volumes of proteasome-glo reagent to
samples (i.e., add 100µl reagent to 100µl sample)
Incubate for 1hr at RT protected from light
Measure LUMINESCENCE
o Remove all filters from the plate reader
o Want to detect all the light produced during the reaction
o Set the Gain (manual) to 100
NOTE: The susbtrate containing reagent can be stored at 4ºC for one month.