A GST-fused protein in vitro PARylation assay was set up by modifying the method provided by the HT Universal Chemiluminescent PARP Assay kit (Trevigen, Gaithersburg, MD, USA). Briefly, GST and GST-fused proteins immobilized on 25 μl of glutathione Sepharose 4B were incubated with recombinant PARP enzyme and PARP cocktail at room temperature for 1 h. After three washes with Nonidet P-40 lysis buffer, the bound proteins were analysed by immunoblotting.
Ht universal chemiluminescent parp assay kit
Manufactured by Bio-Techne
Sourced in United States
The HT Universal Chemiluminescent PARP Assay kit is a laboratory equipment used to detect and quantify the activity of the enzyme Poly(ADP-ribose) Polymerase (PARP) in cell-based and biochemical samples. The assay employs a chemiluminescent detection method to measure PARP activity, providing a sensitive and high-throughput platform for researchers.
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5 protocols using ht universal chemiluminescent parp assay kit
1
In Vitro PARylation Assay for GST-Fused Proteins
2
PARP Inhibition Assay in A549 and H1299 Cells
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The inhibition of PARP was analyzed in A549 and H1299 cells using the HT Universal Chemiluminescent PARP Assay Kit (Trevigen) according to the manufacturer's instructions. Briefly, cells were treated with DMSO or 1 μmol/L niraparib for 15, 30, 60, or 120 minutes, trypsinized, and transferred to a pre-chilled tube. The cells were washed twice with ice cold PBS and resuspended in cold PARP extraction buffer. The cell suspensions were incubated on ice for 30 minutes with periodic vortexing to disrupt the cell membrane. The suspensions were centrifuged and the supernatant transferred to a pre-chilled tube on ice. The histone coated wells of the 96-well plate were rehydrated with 1X PARP buffer and incubated at room temperature for 30 minutes. The PARP buffer was removed and 20 μg of protein as determined by the Bio-Rad Protein Assay was added to each well followed by diluted PARP-HSA enzyme and 1X PARP buffer. The strip wells were then incubated at room temperature for 60 minutes, washed twice with PBS containing 0.1% Triton X-100, and then washed with PBS. Diluted Strep-HRP was added to the strip wells and incubated for 60 minutes at room temperature. The wells were washed again as before. Equal volumes of PeroxyGlow A and B were combined and added to the wells and chemiluminescent readings were obtained immediately using a plate-reader.
3
PARP Inhibition Assay for Exemplary Compounds
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Example 5
Exemplary compounds of the present invention were assayed for inhibitory activity on cell-cycle progression using a PARP assay. The assay was run by an outside analytical laboratory using the standard chemiluminescent PARP inhibition protocol. In addition to the standard controls utilized in this assay, a standard solution of 30 μg minocycline was used as an internal positive control for the test compounds, with the control samples being plated in triplicate.
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4
PARP-1 Activity Determination in iPSCs
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The activity of PARP-1 was determined following the procedure of the HT Universal Chemiluminescent PARP Assay Kit (Trevigen, Cat. No. 4677-096-K). Briefly, approximately 2 × 106 iPS cells were centrifuged at 400×g for 10 min at 4 °C. The supernatant was discarded and the pellet was suspended in 5–10 pellet volumes of cold 1× PARP buffer containing 0.4 mM PMSF, other protease inhibitors, 0.4 M NaCl, and 1% Triton X-100. The cell suspensions were incubated at 4 °C, with periodic vortexing for 30 min.
The disrupted cell suspensions were centrifuged at 10,000×g for 10 min at 4 °C to remove insoluble material. Forty-microliter aliquots of the supernatant were taken, and 10 μl of the poly (ADP-ribose) glycohydrolase (PARG) inhibitor ADP-HPD (Calbiochem, Cat. No. 118415, 60 μg) was added to each aliquot result for a final concentration of 0.2 M ADP-HPD, and the PARP activity was measured. We had examined appropriate concentrations for PARG inhibition in advance, at 0.05, 0.1, 0.2 and 0.25 M, and determined that 0.2 M would be used for the experiment (data not shown). At least 20 μg of protein per well was used in the assay. All samples in 96-well plates were read using a Wallac ARVO SX 1420 Multilabel Counter (PerkinElmer).
The disrupted cell suspensions were centrifuged at 10,000×g for 10 min at 4 °C to remove insoluble material. Forty-microliter aliquots of the supernatant were taken, and 10 μl of the poly (ADP-ribose) glycohydrolase (PARG) inhibitor ADP-HPD (Calbiochem, Cat. No. 118415, 60 μg) was added to each aliquot result for a final concentration of 0.2 M ADP-HPD, and the PARP activity was measured. We had examined appropriate concentrations for PARG inhibition in advance, at 0.05, 0.1, 0.2 and 0.25 M, and determined that 0.2 M would be used for the experiment (data not shown). At least 20 μg of protein per well was used in the assay. All samples in 96-well plates were read using a Wallac ARVO SX 1420 Multilabel Counter (PerkinElmer).
5
In Vitro GST-Fused Protein PARylation Assay
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A GST-fused protein in vitro PARylation assay was set up by modifying the method provided by the HT Universal Chemiluminescent PARP Assay kit (Trevigen, Gaithersburg, MD, USA) [14 (link)]. Briefly, GST and GST-fused proteins immobilized on glutathione Sepharose 4B were incubated with recombinant PARP enzyme and PARP cocktail at room temperature for 1 h. After three washes with Nonidet P-40 lysis buffer, the bound proteins were analysed by western blotting.
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