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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.


BRIEFLY...

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)

     wash w/PBS

     add 100µL GAM-HRP

     wash w/PBS

     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

#K611 Histone H4 96-well plate


Our product areas > Poly/mono ADP-ribose Products > PARP1 Activity Assay Protocol > NEXT

#1020 Anti-pADPR, clone 10H


#2090 PARP1 enzyme


PARP1 DNA activation
3-AB inhibition
Microplate strips

PARP1 Activity Assay Protocol Download

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.

Olaparib PARP1 inhibition

Please contact us with your comments and suggestions about the PARP1 Activity Assay.

And, please inquire about custom configuration and supply of the assay components.