Parp apoptosis colorimetric assay kit

The activities of caspase-9 and caspase-3 were determined using the caspase colorimetric test kits based on spectrophotometric detection of the chromophore p-nitroanilide (p-NA) as a result of the cleavage of the labeled substrate acetyl-Leu-Glu-His-Asp-pNA and Asp-Glu-Val-Asp-pNA, respectively. The assay kits for caspases-9 (Cat. # ab65608) and caspase-3 (Cat. # ab39401) were purchased from Abcam plc. (Cambridge, MA, USA). Free pNA was identified with a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA) at 405 nm.
The PARP/Apoptosis colorimetric assay kit (R&D Systems, Minneapolis, MN, USA; Cat. # 4684-096-K) was used for the determination of PARP activity based on a semi-quantitative measurement of the amount of poly (ADP-ribose) deposited on the immobilized histone proteins. The absorbance values at 450 nm were obtained from a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA). All values were compared to the controls treated by the vehicle.

The caspases-9 activity was determined using a caspase colorimetric test kit (Abcam plc., Cambridge, MA, USA; Cat. # ab65608) based on the spectrophotometric detection of the chromophore p-nitroanilide (pNA) after caspase separation of the substrate labelled acetyl-Leu-Glu-His-Asp-pNA. Caspase-3 colorimetric activity assay kit (Abcam plc., Cambridge, MA, USA; Cat. # ab39401) was used to measure caspase-3 activity according to the spectrophotometric detection of pDNA following the cleavage of the labeled substrate Asp-Glu-Val-Asp-pNA. The free pNA was quantified using a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA) at 405 nm.
PARP activity levels have been measured by PARP/Apoptosis colorimetric Assay Kit (R&D Systems, Minneapolis, MN, USA; Cat. # 4684-096-K) as per the manufacturer’s protocol. The activity of PARP-1 was assessed by semi-quantitative measurement of the amount of poly (ADP-ribose) deposited on immobilized histone proteins and detection of absorbance values at the 450 nm wavelength using a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA). All the values were compared with those obtained from the vehicle-treated control.

The levels of PARP activity were measured by ELISA (PARP/Apoptosis Colorimetric Assay Kit, catalog4684-096-K, R&D Systems) according to the manufacturer’s protocol. A total of 5 × 10⁴ cells/200 μL fresh medium/well in a 96-well, flat-bottom plate were incubated 24 hours for ER stressors or 10 mM etoposide. Samples of 25 μL containing 200 μg proteins were evaluated in triplicate. PARP activity in samples was evaluated by semiquantitatively detecting PAR deposited onto immobilized histone proteins in a 96-well format. An anti-PAR monoclonal antibody, goat anti-mouse IgG-HRP conjugate (both from the kit), and HRP substrate were used to generate a colorimetric signal (450 nm). Absorbance correlates with PARP activity.