Universal colorimetric parp assay kit

The Universal PARP Colorimetric Assay Kit (Trevigen^® Inc., Gaithersburg, MD, USA) was used in order to evaluate the inhibition effect of the tested chemical compounds on PARP1/2 activity. PARP enzymatic activity was assayed according to the manufacturer’s instructions and as previously described [31 (link),32 (link)]. The method is based on the incorporation of biotinylated poly (ADP-ribose) (PAR) onto histone proteins in a 96-stripwell microplate format. Colorimetric readouts at 450 nm absorbance of the PARP activity in a serial of PARP dilutions (units/well) were performed, and a standard curve was prepared (Figure 2). 3-Aminobenzamide (3-AB), a well-known inhibitor of PARP enzymes, was used comparatively as a positive control for testing the inhibitory activity on PARP. The compounds ASA-A, ASA-B, ASA-C and POPA were tested in comparison to the standard well-established PARP inhibitor 3-AB at concentrations of 1–300 μM, for the inhibition of PARP activity (0.6 Unit/well PARP-HSA Enzyme). The tested compounds were loaded in quadruplicate into a 96-well plate coated with histones and biotinylated poly ADP-ribose and allowed to incubate for 1 h. Then, they were treated with horseradish peroxidase (HRP) conjugated streptavidin (strep-HRP) and read in a spectrophotometer at 450 nm [25 (link),33 (link)].