●PARP1 Histone H4 Activity Assay Protocol
Description: The PARP1 Histone H4 Activity Assay Protocol describes a colorimetric assay based upon the PARP1 enzyme PARylation of histone H4 on coated 96-well microplates. The assay is generally easy to perform, low cost, and useful for screening PARP1 inhibitors.
To wells of a histone H4-coated microplate (Cat. #K611):
add 12.5µL test sample (eg: inhibitor)
add 12.5µL PARP1 (Cat. #2090)/activated DNA
Initiate the PARylation:
add 25µL NAD (10 or 100µM)
then incubate for 30min
Stop the rxn:
wash microplate w/PBS
ELISA/colorimetric development of microplate:
add 100µL anti-pADPr, clone 10H (Cat. #1020)
add 100µL GAM-HRP
Add 100µL TMB, set for 15min
Stop w/100µL 0.2N HCl
Measure OD450nm in microplate reader:
OD450 is proportional to the PARylation of
histone H4 in the sample well.
Please see the following publications:
Kotova E, Pinnola AD, Tulin AV. 2011. doi:10.1007/978-1-61779-270-0_29.
Kotova E and Tulin AV. 2017. doi:10.1007/978-1-4939-6993-7_19.
Supplied as: Freely distributed protocol.
Price: The following assay components are available from Tulip Biolabs (other required materials are listed in the protocol):
PARP1 Activity Assay
PARP1 Histone H4 microplate assay was performed at 10µM NAD (top) and 100µM NAD (bottom) with increasing concentrations of Olaparib (note the concentration scale difference). The IC50 for Olaparib at 10µM NAD is 7.47nM, and at 100µM is 55.62nM. Thus, the Olaparib inhibition is competitive with NAD.