ADP-ribose-pNP

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Physical properties

Molecular weight	724.37
Solvent	Water

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501

Overview SDS Protocol

Molecular weight

724.37

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.

Platform

Absorbance microplate reader

Absorbance	405 nm
Recommended plate	Solid white

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		=

Images

Figure 1. Chemical structure for ADP-ribose-pNP

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

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

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

Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein
Authors: Galletto R, Rajendran S, Bujalowski W.
Journal: Biochemistry (2000): 12959

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

Effects of caffeine and adenine nucleotides on Ca2+ release by the sarcoplasmic reticulum in saponin-permeabilized frog skeletal muscle fibres
Authors: Duke AM, Steele DS.
Journal: J Physiol (1998): 43

Adenine nucleotides regulate ADP-ribosylation of membrane-bound actin and actin-binding to membranes
Authors: Schroeder P, Just I, Aktories K.
Journal: Eur J Cell Biol (1994): 3

Trimeric G-proteins of the trans-Golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules
Authors: Barr FA, Leyte A, Mollner S, Pfeuffer T, Tooze SA, Huttner WB.
Journal: FEBS Lett (1991): 239

Inhibition and labeling of the Ca2(+)-ATPase from sarcoplasmic reticulum by periodate oxidized ATP
Authors: Mignaco J, Scofano HM, Barrabin H.
Journal: Biochim Biophys Acta (1990): 305

Documents

Safety data sheet
Product protocol
Certificate of analysis

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