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Index / Trevigen / Universal Colorimetric PARP Assay Kit w_ Histone Coated Strip Wells / Product Detail : 4677-096-K Universal Colorimetric PARP Assay Kit w_ Histone Coated Strip Wells
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#4677-096-K Universal Colorimetric PARP Assay Kit w_ Histone Coated Strip Wells

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Product name : Universal Colorimetric PARP Assay Kit w_ Histone Coated Strip Wells

Catalog number : 4677-096-K

Quantity: 96 Samples

Availability: Yes

Supplier name : Trevigen

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4677-096-K Universal Colorimetric PARP Assay Kit w/ Histone Coated Strip Wells

Colorimetric assay kit for candidate inhibitor screening and determination of IC50 values of PARP inhibitors.

I. Introduction
Poly ADP-ribosylation of nuclear proteins is a post-translational event that occurs in response to DNA damage. Poly (ADP-ribose) polymerase (PARP) catalyzes the NAD-dependent addition of poly (ADP-ribose) to itself and adjacent nuclear proteins such as histones. PARP contributes to the sequence of events that occurs during DNA base excision repair.1 Whereas PARP-mediated induction of necrosis can occur by extensive depletion of the intracellular NAD pool,2 the cleavage of PARP-1 promotes apoptosis by preventing DNA repair-induced survival and by blocking energy depletion-induced necrosis.3 Experimental models have shown that PARP inhibition prevents tissue damage in animal models of myocardial and neuronal ischemia, diabetes, septic shock, and vascular stroke.4-11 Moreover, PARP inhibition promotes chemosensitization and radiosensitization of tumors.12 Trevigen's HT Universal 96-well PARP Assay Kits measure the incorporation of biotinylated poly(ADP-ribose) onto histone proteins in a 96-well strip well format. This assay is ideal for the screening of PARP inhibitors and determining IC50 values. Important features of the assay include: 1) colorimetric, nonradioactive format; 2) higher throughput 96 test size; and 3) sensitivity down to 0.01 Units of PARP per well. Histone-coated 96-well clear strip wells (4677-096- P) are available separately for your convenience.

II. Precautions and Limitations
1. For Research Use Only. Not for use in diagnostic procedures.
2. The physical, chemical, and toxicological properties of the chemicals and reagents contained in the HT Universal Colorimetric PARP Assay Kit may not yet have been fully investigated. Therefore, Trevigen recommends the use of gloves, lab coats, and eye protection while using any of these chemical reagents. Trevigen assumes no liability for damage resulting from handling or contact with these products. MSDS are available on request.

III. Materials Supplied


IV. Materials/Equipment Required But Not Supplied Reagents:
1. Inhibitors
2. PBS (cat# 4870-500)
3. PBS + 0.1% Triton X-100
4. Distilled water
5. 0.2N HCl or 5% Phosphoric acid

1. 1 - 200 μl and 100-1000 μl pipette tips

1. Micropipettes
2. Multichannel pipettor 10 - 200 μl
3. Wash bottle or microstrip wells washer (optional)
4. 96-well plate reader with 450 nm filter

V. Reagent Preparation
1. 10X Strep-Diluent. This solution is used as a diluent for the Strep-HRP. Dilute 1:10 with dH20 before use.
2. 20X PARP Buffer. Dilute the 20X PARP Buffer to 1X (1:20) with dH2O. The 1X PARP Buffer is used to rehydrate the histone coated wells, and to dilute the enzyme, PARP Cocktail, and the inhibitors to be tested.
3. 10X PARP Cocktail. Dilute the 10X PARP Cocktail as follows: 10X PARP Cocktail (Cat# 4671-096-03) 2.5 μl/well 10X Activated DNA (Cat# 4671-096-06) 2.5 μl/well 1X PARP Buffer 20 μl/well
4. PARP Enzyme. The kit contains 50 μl of PARP-HSA enzyme at a concentration described in the enclosed Product Data Sheet. The enzyme should be diluted appropriately with 1X PARP Buffer just before use. Note: Diluted enzyme should be used immediately and any remainder discarded.
5. PARP Inhibitors. The 3-aminobenzamide (3-AB) is provided at 200 mM in ethanol as a control inhibitor. 3-AB will inhibit the activity of PARP at a wide range of concentrations from 2 μM to 10 mM. Serially dilute the stock 3-AB or your PARP inhibitor(s) with 1X PARP Buffer and add to designated wells.
6. Strep-HRP. Just before use, dilute Strep-HRP (cat# 4800-30-06) 500-fold with 1X Strep- Diluent (cat# 4671-096-04). A total of 50 μl/well of diluted Strep-HRP is required in the assay.
7. TACS-Sapphire™. Prewarm TACS-Sapphire to room temperature before use. TACS-Sapphire is a colorimetric substrate that turns blue in the presence of Horseradish Peroxidase (HRP). The addition of an equal volume of 0.2 M HCl or 5% phosphoric acid stops the reaction to generate a yellow color stable for up to 60 minutes that can be read at 450 nm.

VI. PARP Inhibitor Assay Protocol
A. Ribosylation Reaction

Note: Do not premix the PARP-HSA enzyme and the PARP Cocktail since PARP will autoribosylate in the presence of NAD.
1. Remove strip wells from the wrapper and add 50 μl/well of 1X PARP Buffer to rehydrate the histones. Incubate at room temperature for 30 minutes. Remove the 1X PARP Buffer from the wells by tapping the strip wells on paper towels.
2. Add serial dilutions of inhibitor of interest (prepared in Section V.5) to appropriate wells.
3. Add diluted PARP enzyme (0.5 Unit/well prepared in Section V.4) to the wells containing inhibitor. Incubate for 10 minutes at room temperature.
4. Controls:
i. Negative Control: A negative control without PARP should be prepared to determine background absorbance.
ii. Activity Control for PARP Inhibitor Study: 0.5 Unit/ well PARP-HAS without inhibitors. These wells provide the 100% activity reference point.
iii. Optional PARP Standard Curve: Serially dilute the PARP-HSA standard in cold microtubes with 1X PARP Buffer such that the total activity is 1 Unit/25 μl, 0.5 Units/25 μl, 0.25 Units/25 μl, 0.1 Units/25 μl, 0.05 Units/25 μl, 0.025 Units/25 μl, and 0.01 Units/25 μl. Add 25 μl of each standard to triplicate wells.
5. Distribute 25 μl of 1X PARP Cocktail into each well using a multichannel pipettor.
6. The final reaction volume is 50 μl:
i. PARP Inhibitor Study:
Volume Order of Addition Diluted test inhibitor or 1X PARP buffer X μl 1 Diluted PARP-HSA enzyme (0.5 Unit) Y μl 2 1X PARP cocktail 25 μl 3
Total volume 50 μl Where X + Y = 25 μl
Note: If X = 10 μl, make the concentration of your inhibitor 5-fold that of the final inhibitor concentration in the reaction since the reaction volume is 50 μl. In this example, Y = 15 μl. Therefore, dilute the PARP-HSA enzyme to 0.5 units/15 μl in 1X PARP Buffer.
ii. Optional PARP Standard Standard Volume Order of Addition Diluted PARP Standards 25 μl 1 1X PARP cocktail 25 μl 2 Total volume 50 μl
7. Incubate the strip wells at room temperature for 60 minutes.

B. Detection
1. Wash strip wells 2 times with 1X PBS + 0.1% Triton X-100 (200 μl/well)followed by 2 washes with 1X PBS. Ensure that all the liquid is removed following each wash by tapping strip wells onto paper towels.
2. Add 50 μl per well of diluted Strep-HRP (prepared in Section V.6). Incubate at room temperature for 60 minutes.
3. Wash strip wells 2 times with 1X PBS + 0.1% Triton X-100 (200 μl/well) followed by 2 washes with 1X PBS. Ensure that all the liquid is removed following each wash by tapping strip wells onto paper towels.
4. Add 50 μl per well of pre-warmed TACS-Sapphire™ colorimetric substrate and incubate, in the dark, for 15 minutes at room temperature. Stop the reactions by adding 50 μl per well of 0.2M HCl or 5% Phosphoric Acid and read the absorbance at 450 nm.
VII. Data Interpretation
Typical colorimetric PARP standard curve and inhibition curves for the PARP inhibitors 3-aminobenzamide (provided in the kit), benzamide and 4-amino-1,8- naphthalimide (available from Trevigen) are graphically represented in Figure 1

VIII. References
1. Golstein P, Kroemer G. 2007. Cell death by necrosis: Towards a molecular definition. Trends Biochem Sci 32: 37-43.
2. D’Amours D, Sallmann FR, Dixit VM, Poirer GG. 2001. Gain-of-function of poly(ADPribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis. J Cell Science 114: 3771-78.
3. Eliasson M, Sampei K, Mandir AJ. 1997. Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia. Nat Med 3:1089- 95.
4. Miller MS, Zobre C, Lewis M. 1993. In vitro neuroprotective activity of inhibitors of poly-ADP ribose polymerase. Soc Neurosci Abstr 19.1656
5. Pieper AA, Verma A, Zhang J, Snyder SH. 1999. Poly(ADP-ribose) polymerase, nitric oxide and cell death. Trends Pharmacol Sci 20:171-81.
6. Thiemermann C, Bowes J, Myint FP, Vane JR. 1997. Inhibition of the activity of poly(ADP-ribose) synthase reduces ischemia-reperfusion injury in the heart and skeletal muscle. Proc Natl Acad Sci USA 94:679-83.
7. Virag L, Szabo C. 2002. The therapeutic potential of Poly(ADPribose) Polymerase inhibitors. Pharmacol Rev 54:375-429.
8. Tong WM, et al. 2002. Synergistic role of Ku80 and poly(ADP-ribose) Polymerase in suppressing chromosomal aberrations and liver cancer formation. Cancer Res. 62:6990-6.
9. Kim MY, Mauro S, Gevry N, Lis JT, Kraus WL. 2004. NAD-Dependent Modulation of
Chromatin Structure and Transcription by Nucleosome Binding Properties of PARP-1. Cell 119:803–14.
10. Kauppinen TM, Swanson RA. 2005. Poly(ADP-ribose) polymerase-1 promotes microglial
activation, proliferation inhibitors for cancer therapy. Expert Rev Mol Med. 7:1-20.
11. Curtin NJ. 2005. PARP 1. Satoh MS, Lindahl T. 1992. Role of poly(ADP-ribose) formation in DNA repair. Nature 356: 356–8.

IX. Troubleshooting

XI. Appendix
Reagent composition:

1. 1X PBS (pH 7.4): 7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 145 mM NaCl.
2. 10X Strep Diluent: Biotin-reduced proprietary blocking solution.
3. 20X PARP Buffer: Proprietary buffer solution.
4. 10X PARP Cocktail: Proprietary solution containing biotinylated NAD.
5. PARP-HSA Enzyme: PARP-HSA is provided at a concentration described in the enclosed Product Data Sheet.
6. 3-Aminobenzamide: 200 mM 3-aminobenzamide in Ethanol.
7. TACS-Sapphire™ (cat# 4822-96-08): Peroxidase substrate readable at 630 nm (blue) or at 450 nm (yellow) after stopping reaction with 0.2 M HCl or 5% Phosphoric Acid.
8. 10X Activated DNA: Activated Herring Sperm DNA in 10 mM Tris-Cl (pH 8.0), 1 mM EDTA.
9. Strep-HRP: Provided at 500X Concentration
The product accompanying this document is intended for research use only and is not intended for diagnostic purposes or for use in humans.

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