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ADP-ribose-pNP
ADP-ribose-pNP is a colorimetric substrate for assessing activity of poly(ADP-ribose)polymerase (PARP) enzymes. The absorbance of released p-nitrophenol is determined at 405 nm, and the slope of the calibration curve is used to convert the absorbencies to moles of product generated. With ADP-ribose-pNP as the colorimetric substrate, PARP-1 was determined to have the largest Km and Vmax values (151 uM and 1.30 nmolmin-1mg-1 respectively) followed by tankyrase-1 (82 uM and 18 pmolmin-1mg-1 respectively) and VPARP (46 uM and 2 pmolmin-1mg-1 respectively). This colorimetric substrate can be used to determine the kinetic parameters for PARP-1, tankyrase-1, and VPARP, and to screen small-molecule inhibitors of PARP-1, tankyrase-1, and VPARP. ADP-ribose-pNP-based continuous assay has considerable advantages over standard discontinuous PARP assays, enabling the high throughput screening of PARP-1, tankyrase-1, and VPARP activities and their inhibitors.
Example protocol
AT A GLANCE
Important notes
The following recommended procedure can be adapted for measuring PARP-1, tankyrase-1, and VPARP activities and their inhibitors. The optimum conditions must be determined experimentally for each test.
PREPARATION OF STOCK SOLUTION
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
1. ADP-ribose-pNP stock solution:
Make 5 - 10 mM stock solution in H2O. Note: The stock solution should be used promptly.
PREPARATION OF WORKING SOLUTION
ADP-ribose-pNP working solution:
Prepare 0.25 mM assay solution by diluting the stock solution with assay buffer (50mM Tris, 10mM MgCl2, pH 8.0).
SAMPLE EXPERIMENTAL PROTOCOL
- Add 0.01 mL/well of sample solution into 0.09 mL/well assay solution to make a final volume of 0.1 mL in a 96-well clear plate.
- Monitor the plate using an absorbance microplate reader at 405 nm.
Calculators
Common stock solution preparation
Table 1. Volume of Water needed to reconstitute specific mass of ADP-ribose-pNP to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 138.051 µL | 690.255 µL | 1.381 mL | 6.903 mL | 13.805 mL |
| 5 mM | 27.61 µL | 138.051 µL | 276.102 µL | 1.381 mL | 2.761 mL |
| 10 mM | 13.805 µL | 69.025 µL | 138.051 µL | 690.255 µL | 1.381 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
References
View all 9 references: Citation Explorer
A colorimetric substrate for poly(ADP-ribose) polymerase-1, VPARP, and tankyrase-1
Authors: Nottbohm AC, Dothager RS, Putt KS, Hoyt MT, Hergenrother PJ.
Journal: Angew Chem Int Ed Engl (2007): 2066
Authors: Nottbohm AC, Dothager RS, Putt KS, Hoyt MT, Hergenrother PJ.
Journal: Angew Chem Int Ed Engl (2007): 2066
Poly(ADP-ribose) polymerase as a key player in excitotoxicity and post-ischemic brain damage
Authors: Meli E, Pangallo M, Baronti R, Chiarugi A, Cozzi A, Pellegrini-Giampietro DE, Moroni F.
Journal: Toxicol Lett (2003): 153
Authors: Meli E, Pangallo M, Baronti R, Chiarugi A, Cozzi A, Pellegrini-Giampietro DE, Moroni F.
Journal: Toxicol Lett (2003): 153
Pharmacological identification of P2X1, P2X4 and P2X7 nucleotide receptors in the smooth muscles of human umbilical cord and chorionic blood vessels
Authors: Valdecantos P, Briones R, Moya P, Germain A, Huidobro-Toro JP.
Journal: Placenta (2003): 17
Authors: Valdecantos P, Briones R, Moya P, Germain A, Huidobro-Toro JP.
Journal: Placenta (2003): 17
Kinetic mechanism of nucleotide cofactor binding to Escherichia coli replicative helicase DnaB protein. stopped-flow kinetic studies using fluorescent, ribose-, and base-modified nucleotide analogues
Authors: Bujalowski W, Jezewska MJ.
Journal: Biochemistry (2000): 2106
Authors: Bujalowski W, Jezewska MJ.
Journal: Biochemistry (2000): 2106
Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein
Authors: Galletto R, Rajendran S, Bujalowski W.
Journal: Biochemistry (2000): 12959
Authors: Galletto R, Rajendran S, Bujalowski W.
Journal: Biochemistry (2000): 12959
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