Navigation Links
A DELFIA Assay for Inhibition of PARP

The DELFIA time-resolved fluorescence assay for inhibition of poly(ADP-ribose) polymerase (PARP) is a 96-well microplate assay that measures the inhibition of PARP-activated incorporation of biotinylated nicotinamide adenine diphsophate (NAD). The incorporation is detected using Europiumlabelled streptavidin. The method is a convenient way to screen test compounds for PARP activity in high throughput primary screening and secondary screening.

Poly(ADP-ribose) polymerase (PARP) is a nuclear protein involved in the response to DNA damage, where it catalyzes the polymerization of nicotinamide adenine diphsophate (NAD) into chains of poly(ADPribose) polymers1. The polymerization occurs on a number of nuclear proteins, as well as on the PARP protein itself. Following activation by DNA strand breaks, PARP hydrolyzes NAD and catalyzes the formation of PARP onto itself and other nuclear proteins, with the release of nicotinamide. PARP binds to singleand double-stranded DNA breaks. When the enzyme undergoes auto-poly (ADPribosyl) ation, the automodified PARP dissociates from the DNA and becomes inactive2.

Inhibitors of PARP have been shown to help prevent ischemic injury in the brain, increase apoptosis in cancer cells, prevent infiltration of neutrofils and subsequent inflammation, and to suppress the production of nitric oxide synthase in macrophages3,4. In response to low to moderate levels of DNA damage, PARP is activated and NAD levels in the cells decrease. Experiments with PARP inhibitors suggest that PARP activity may be necessary to rescue cells after low to moderate levels of DNA damage. In contrast, when cells experience massive levels of DNA damage and DNA strand breaks, activation of PARP can lead to depletion of NAD and ATP, resulting in a marked decrease in energydependent processes and in DNA repair. In this situation, activation of PARP by massive DNA damage processes may actually be a suicide response, since it causes rapid NAD and ATP depletion and leads to cell death before the cell can repair the DNA damage.

METHODS

The DNA used in the polymerization reaction was fragmented by hydrodynamic shearing. The DNA was passed through the orifice of a 28-gauge hypodermic needle. This method of shearing generates 1.5-2.0 kb DNA fragments5. The polymerization reaction was performed in an uncoated 96-well microtiter plate. To the well was added: 2.5 μg histones (Trevigen/Upstate U.C. Biotechnology), 1 μg DNA (Sigma), 15 μM NAD + Bio-NAD (Trevigen) in a 17:1 or 1:1 ratio, 1 mM 3- aminobenzamide6 (inhibitor of PARP, Sigma) 0.5 U PARP enzyme (Trevigen) and PARP buffer (500 mM Tris-HCl, 250 mM MgCl2). The reaction was performed at room temperature, for 30 minutes. The plate was then washed 2 times using the DELFIA Platewash. Eu-labelled streptavidin was diluted in DELFIA Assay Buffer to 200 ng/ well and incubated for 30 minutes at room temperature. The plate was washed 4 times using the DELFIA Platewash, prior to addition of 200 μL/well DELFIA Enhancment Solution. The fluorescence was measured using VICTOR2 TM plate reader.

The data shows that the DELFIA timeresolved fluorescence assay for inhibition of PARP allows for the measurement of the incorporation of biotinylated NAD. This technology is therefore suitable for the screening of inhibitors of PARP where the formation of poly(ADP-ribose) chains is inhibited.

REFERENCES:

1. Satoh, M. S. and Lindahl, T. 1994. Role of poly(ADP-ribose) formation in DNA repair. Nature 356, 356-358

2. Lautier, D., Langeux, J., Thibodeau, L., Menard, L. and Poirier, G. G. 1993. Molecular and biochemical features of poly(ADP-ribose) metabolism. Mol Cell Biochem 122, 171-193

3. Thiermann, C., Bowes, J., Myint, F. P. and Vane, J. R. 1997. Inhibition of the activity of poly(ADP ribose) synthease reduces ischemia-reperfusion injury in the heart and skeletal muscle. Proc. Natl. Acad. Sci. USA 94, 679-683

4. Heller, B., Wang, Z-Q., Wagner, E. F., Radons, J., Brkles, A., Fehsel, K., Burkart, V. and Kolb, H. 1995. Inactivation of the poly(ADP-ribose) polymerase gene affects oxygen radical and nitric oxide toxicity in islet cells. J. Biol. Chem. 270, 11176-11180

5. Davidson, P. F. 1959. The effect of hydrodynamic shear on the deoxyribonucleic acid from T2 and T4 bacteriophages. Proc. Natl. Acad. Sci. 45, 1560-1568

6. Purnell, M. R. and Whish, W. J. 1980. Novel inhibitors of poly(ADP-ribose) synthetase. Biochem. J . 185, 775-777


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. DELFIA Protein Kinase assays.
2. A Positive Selection Assay for Mutation Analysis in Big Blue Animals
3. Luminol-enhanced Assay for Superoxide Anion (O2 face="Symbol" )
4. Organize Your Data from the Big Blue Mutation Assay
5. Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations
6. RNase Protection Assay (RPA) Using DIG-Labeled RNA Probes
7. measuring cell proliferation with SpectraMax M5, M2 and Gemini microplate readers using the CyQUANT Cell Proliferation Assay
8. Matched siRNAs and Assays: Ambion + Applied Biosystems = RNAi Success
9. Using Validated siRNAs in Functional Genomic Assays
10. Nuclease Protection Assays: The Basics
11. RNArticles: RNA Amplification Used to Develop a New In Vitro HCMV Assay
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: DELFIA Assay for Inhibition PARP

(Date:8/22/2014)... Albany, New York (PRWEB) August 22, 2014 ... represent one of the interesting and challenging endeavors ... eye renders highly impervious properties to the foreign ... such as possibility of self medication and minimal ... of low molecular weight drugs and provides specific ...
(Date:8/22/2014)... Los Angeles (PRWEB) August 22, 2014 ... for age-defying products, is in the spotlight again. ... night cream and its NeriumFirmTM Body Contouring Cream ... presented by Backstage Creations during the 66th Primetime ... the patented NAE-8® extract, third-party clinical trials show ...
(Date:8/21/2014)... the Institute for Molecular Medicine Finland (FIMM), University ... a novel "man and machine" decision support system ... was described in PLOS One scientific ... on computer vision algorithms similar to those used ... only the diagnostically most relevant areas. Tablet computers ...
(Date:8/21/2014)... 21, 2014 Telomere Biosciences, LLC (Telomere ... "TELO-20 for Dogs" with Telomerase Activation Complex ... the world for dogs. Telomeres are the protective ... the body. A wealth of ground-breaking research by ... Science and Aging, including Nobel laureates, has demonstrated ...
Breaking Biology Technology:Ocular Drug Delivery Technologies Market: Global Industry Analysis, Size, Share, Growth, Trends and Forecast 2014 - 2020 2Ocular Drug Delivery Technologies Market: Global Industry Analysis, Size, Share, Growth, Trends and Forecast 2014 - 2020 3Ocular Drug Delivery Technologies Market: Global Industry Analysis, Size, Share, Growth, Trends and Forecast 2014 - 2020 4Nerium International Joins Television’s Biggest Stars at Backstage Creations Giving Suite at the 66th Primetime Emmy Awards 2Nerium International Joins Television’s Biggest Stars at Backstage Creations Giving Suite at the 66th Primetime Emmy Awards 3A novel 'man and machine' decision support system makes malaria diagnostics more effective 2Telomere Biosciences Announces: TELO-20 for Dogs, the World's First Telomere-Lengthening Supplement for Dogs, to Promote Healthy Aging and Longevity 2Telomere Biosciences Announces: TELO-20 for Dogs, the World's First Telomere-Lengthening Supplement for Dogs, to Promote Healthy Aging and Longevity 3
... of decorative, flower-like defects in the nanomaterial graphene could ... electrical and mechanical properties, according to researchers at the ... Tech. In a new paper,* the team for the ... could occur naturally or be induced to occur in ...
... of the March 11 earthquake and rolling blackouts that ... award-winning undergraduate internship program NanoJapan is scrambling to run ... U.S. undergraduates travel to Japan for lab internships, NanoJapan ... send 25-30 students to Rice University laboratories for three ...
... May 25, 2011 Anadys Pharmaceuticals, Inc. (Nasdaq: ... the treatment of hepatitis C, announced today that it will ... the Company,s oral immuno-modulator that acts via the TLR-7 pathway, ... Meeting in Chicago, IL.  The data from the completed Phase ...
Cached Biology Technology:Defect in graphene may present bouquet of possibilities 2Reverse NanoJapan: Rice to host 25-30 Japanese students 2Anadys Highlights ANA773 Data to be Presented at ASCO 2Anadys Highlights ANA773 Data to be Presented at ASCO 3Anadys Highlights ANA773 Data to be Presented at ASCO 4
(Date:8/21/2014)... Aug. 21, 2014 Nxt-ID, Inc. (OTCQB: NXTD), ... commerce market, announced today that its shares of common ... be issued in its proposed underwritten public offering of ... on The Nasdaq Capital Market, subject to closing of ... and warrants, and will trade under the symbol "NXTD" ...
(Date:8/21/2014)... ubiquitous skin fungus linked to dandruff, eczema and other ... to even further global reachesincluding Hawaiian coral reefs and ... vents. , A review in the scientific journal ... of the fungi of the genus Malassezia ... sequencing datasets from around the world. , University of ...
(Date:8/21/2014)... -- Nine researchers from Beth Israel Deaconess ... in "The World,s Most Influential Scientific Minds 2014," ... Thomson Reuters ScienceWatch, a web resource for science metrics ... recognized as among the most influential "are performing and ... the advancement of their science," according to a Thomson ...
Breaking Biology News(10 mins):Nxt-ID Approved for NASDAQ Capital Market Listing 2Nxt-ID Approved for NASDAQ Capital Market Listing 3From dandruff to deep sea vents, an ecologically hyper-diverse fungus 2BIDMC researchers named among 'the most influential scientific minds' 2BIDMC researchers named among 'the most influential scientific minds' 3
... commercially available mineral waters, the researchers found evidence of ... the water. What,s more, these chemicals are potent in ... in the New Zealand mud snail. These findings, which ... of plastic food packaging materials act as functional estrogens, ...
... diseases that can travel with the wind have the ... understood, according to a new study, in findings that ... but also a new fungus that threatens global wheat ... State University and other institutions, concluded that invading diseases ...
... is available in Spanish . , ... of psychosis and schizophrenia, and those episodes of psychosis ... characteristics, both before their appearance and in the clinical ... conclusions of the doctoral thesis "Neurodevelopment and environmental stress ...
Cached Biology News:Hormone-mimics in plastic water bottles -- just the tip of the iceberg? 2New wheat disease could spread faster than expected 2New wheat disease could spread faster than expected 3Daily consumption of cannabis predisposes to the appearance of psychosis and schizophrenia 2
... The CHEMICON APO-BRDU Kit is ... labeling DNA breaks and total cellular DNA ... The kit contains the instructions and ... including; positive and negative control cells for ...
... overlay agarose, is a low melting point ... to secure IPG strips in place for ... incorporated to allow monitoring of electrophoresis runs. ... melting point agarose in 1x Tris, glycine, ...
... cell is an apparatus dedicated to general ... agarose slab gels. The cell includes the ... tray, 5 glass plates, 50 sheets of ... templates, and instructions. Dimensions are 21 x ...
... range of next-generation features into a small-footprint ... for flexible laboratory solutions. It puts every ... dilution, dispensing, and integrations into a ... flexible as it is efficient and economical. ...
Biology Products: