AP site Detection Principle
Mechanism of ARP Tagging at an Abasic Site
Recent Publications
| Samples from | Treatments | References |
|---|---|---|
| MEF cells | Cisplatin, Oxaliplatin | Novel Role of Base Excision Repair in Mediating Cisplatin CytotoxicityA. Kothandapani, et al., J Biol Chem, 286, 14564(2011) |
| SF767glioblastoma | AP endonuclease repair inhibitor | Novel Small-Molecule Inhibitor of Apurinic/Apyrimidinic Endonuclease 1 Blocks Proliferation and Reduces Viability of Glioblastoma CellsA. Bapat, et al., J Pharmacol Exp Ther, 334, 988(2010) |
| hippocampaltissue | global cerebral ischemia | Apurinic/apyrimidinic endonuclease APE1 is required for PACAP-induced neuroprotection against global cerebral ischemiaR. A. Stetler, et al., PNAS, 107, 3204(2010) |
| CHO cells | dominant-negative form of AP endonuclease 1 expressing | Impairment of APE1 Function Enhances Cellular Sensitivity to Clinically Relevant Alkylators and AntimetabolitesD. R. McNeill, et al., Mol Cancer Res, 7, 897(2009) |
| C57BL/6J mouse | Valsartan | Temporary Pretreatment With the Angiotensin II Type 1 Receptor Blocker, Valsartan, Prevents Ischemic Brain Damage Through an Increase in Capillary DensityJ. Li, et al., Stroke, 39, 2029(2008) |
How to Prepare a Calibration Curve1. Calculate the average O.D. of each ARP-DNA standard solution.2. Subtract the blank O.D. from the average O.D.a)3. Plot the O.D. corresponding to the number of AP sites of the standard solution. X-axis is the number of AP sites and Y-axis is the O.D.4. Determine the number of AP sites in the sample using this calibration curve.
a) The blank O.D. is about 0.04-0.06 and the O.D. of the 40 ARP DNA standard solution is about 0.8-1.0. The O.D. value depends on HRPStreptavidin activity.
Fig. 3 Typical calibration curve of DNA Damage Quantification Kit
1. T. Lindahl, et al., Rate of Depurination of Native Deoxyribonucleic Acid. Biochemistry. 1972;11:3610-3618.2. M. Liuzzi, et al., A New Approach to the Study of the Base-excision Repair Pathway Using Methoxyamine. J Biol Chem. 1985;260:5252-5258.3. A. Sancar, et al., DNA Repair Enzymes. Annu Rev Biochem. 1988;57:29-67.4. M. Weinfeld, et al., Response of Phage T4 Polynucleotide Kinase Toward Dinucleotides Containing Apurinic Sites: Design of a 32P-postlabeling Assay for Apurinic Sites in DNA. Biochemistry. 1990;29:1737-1743.5. B. X. Chen, et al., Properties of a Monoclonal Antibody for the Detection of Abasic Sites, a Common DNA Lesion. Mutat Res. 1992;273:253-261.6. J. A. Gralnick, et al., The YggX Protein of Salmonella enterica Is Involoved in Fe(II) Trafficking and Minimizes the DNA Damage Cause by Hydroxyl Radicals:Residue CYS-7 is Essential for YggX Function. J Biol Chem. 2003;278:20708-20715.7. J. W. Pippin, et al., DNA Damage is a Novel Response to Sublytic Complement C5b-9 Induced Injury in Podocytes. J Clin Invest. 2003;111:877-885.8. S. Watanabe, et al., Methylated DNA-binding Domain 1 and Methylpurine DNA Glycosylase Link Transcriptional Repression and DNA Repair in Chromatin. PNAS. 2003;100:12859-12864.9. M. Endres, et al., Folate Deficiency Increases Postischemic Brain Injury. Stroke. 2005;36:321-325.10. J. Li, et al., Angiotensin II-Induced Neural Differentiation via Angiotensin II Type 2 (AT2) Receptor-MMS2 Cascade Involving Interaction between AT2 Receptor-Interacting Protein and Src Homology 2 Domain-Containing Protein-Tyrosine Phosphatase 1. Mol Endocrinol. 2007;21:499-511.11. D. R. McNeill, et al., A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents. Mol Cancer Res. 2007;5:61-70.12. C. A. Downs, et al., Cellular pathology and histopathology of hypo-salinity exposure on the coral Stylophora pistillata. Sci Total Environ. 2009;407:4838-4851.13. C. A. Downs, et al., Symbiophagy as a cellular mechanism for coral bleaching. Autophagy. 2009;5:211-216.
No, you cannot use this kit to determine the number of abasic sites in single-stranded DNA or RNA. The O.D. reading of single-stranded DNA will be nearly twice that of double-stranded DNA because of the binding efficiency on the microplate.
Prepare a DNA pellet and store at -20°C or -80°C if the DNA cannot be labeled with ARP immediately after isolation. After ARP labeling, the sample can be stored at 4°C in TE Buffer for several months.
You can use general protocols or commercially available DNA isolation kits. Between 2 to 4 abasic sites per 1 x 105 base pairs will be created during the DNA isolation process. Therefore, use the same isolation method to prepare each DNA sample.
You can either use a filtration tube to concentrate your sample DNA or ethanol precipitation to recover DNA as a pellet and then re-dissolve it to prepare a 100 μg per ml solution.
Add the same volume of ARP Solution and follow the manual. The recovery of the ARP-labeled DNA may be lower than the usual reactions, so measure the ARP-labeled DNA solution. The average recovery rate of the 0.5 μg DNA and 0.25 μg DNA is 70% and 50%, respectively.
Dojindo细胞分析
细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。
WST检测机制
ß-半乳糖苷酶检测试剂
细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢
| 应用 | 产品展示 |
| 细胞生长检测，药物筛选，比色/荧光检测 | 细胞计数试剂盒-8 细胞计数试剂盒8 + 96孔有机硅定向剂 细胞计数试剂盒-F 细胞毒性LDH检测试剂盒 -WST 96孔有机硅定向剂 MTT |
| 了解检测机制的差异： | 点击这里 |
| 细胞周期分析 | 细胞周期测定溶液深红色 细胞周期测定溶液蓝色 |