| Assay Method Information | |
| | PARG Activity Assay |
| Description: | PARG Activity Assay was performed in Streptavidin-coated 96-well plates. Terminal-biotinylated poly (ADP-ribose) polymer was diluted to a final concentration of 25 nM with 0.2% bovine serum albumin (BSA) containing phosphate-buffered saline solution (PBS) and used for the substrate solution. The substrate solution was added to the 96-well plate at the volume of 200 micro liter per well and incubated for 3 hours at room temperature. After the incubation, the substrate solution was discarded and assay plate was washed 4 times with 220 micro liter of PBS containing 0.1% Tween20 (PBST). Recombinant human PARG protein was diluted with 0.2% BSA containing PBS to a final concentration of 0.0185 nM. The diluted enzyme solution was pre-incubated with compounds for 1 hour at room temperature in a 96-well plate and transferred 100 micro liter per well to the poly (ADP-ribose) substrate-coated assay plate. The plate was incubated for 1 hour at 25° C. and then washed 4 times with 220 micro liters of PBST. For the detection of non-digested poly (ADP-ribose) polymer on the plate, mouse anti-poly (ADP-ribose) antibody diluted with 0.2% BSA containing PBS was added 100 micro liters per well and incubated 1 hour at room temperature. Plate was washed 4 times with TBST and 100 micro liter of HRP-labeled anti-mouse Ig antibody was added to the wells. Plate was incubated for 30 minutes at room temperature and then washed 4 times with PBST. For the detection 100 micro liters of TMB substrate solution was added to the wells. After sufficient color development, reaction was stopped by addition of 50 micro liter of 0.2 M Sulfonic acid. |
| Affinity data for this assay | |
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