500095 | OxyDNA Assay Kit, Fluorometric

500095
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          描述
          概述 Assay kit used to detect oxidative damage to DNA in vitro. Based on the direct binding of a fluorescent probe to the DNA adduct 8-oxoguanine, a major oxidation product and an important indicator of free radical-induced DNA damage and oxidative stress. The FITC-conjugate binds to 8-oxoguanine in damaged cells and fluorescence is monitored using flow cytometery (excitation 495 nm, emission 515 nm).
          产品目录编号 500095
          品牌系列 Calbiochem®
          参考
          参考 Silva, P.F.N. et al. 2007. Theriogenology 67, 609.
          Babbar, N. and Casero, Jr., R.A. 2006. Cancer Res. 66, 11125.
          Lai, K.N., et al. 2006. Nephrol. Dial. Transplant. 21, 1188.
          Sampson, M.J. et al. 2006. Diabetes Care 29, 283.
          Maniscalco, W.M., et al. 2005. Pediatric Res. 58, 594.
          Osella, D., et al. 2005. Inorganica Chimica Acta 358, 1993.
          Adamek B et al. 2004. Polish Journal of Environmental Studies 13 Suppl. II, 29.
          Roper, J.M., et al. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045.
          Ogawa, Y. et al. 2003. International Journal of Molecular Medicine 11, 27.
          Ogawa, Y. et al. 2003. International Journal of Molecular Medicine 11, 149.
          Sparrow J.R. et al. 2003. Investigative Ophthalmology and Visual Science (14)5, 2245.
          Tao Peng, H-M. et al. 2003. World J Gastroenterol 9, 2186.
          Riemschneider, S., et al. 2002. Acta Derm. Venereol. 82, 325.
          de Zwart, L.L., et al. 1999. Free Radic. Biol. Med. 26, 202.
          Tsurudome, Y., et al. 1999. Carcinogenesis 20, 1573.
          Inoue, M., et al. 1998. Jpn. J. Cancer Res. 89, 691.
          Kasai, H. 1997. Mutat. Res. 387, 147.
          Cooke, C., et al. 1996. Toxicol. Ecotoxicol. News 3, 101.



          Selected Citations
          Roper, J. M., et al. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045.
          Babbar, N., and Casero, R. A., Jr., 2006. Cancer Res. 66, 23.


          产品信息
          检测方法 Flow cytometry or fluorescence microscopy
          形式 50 Tests
          试剂盒包含 Wash Concentrate, Binding Protein-FITC Conjugate, and a user protocol.
          应用
          应用参考 Fluorescent Microscopy Lai, K.N., et al. 2006. Nephrol. Dial. Transplant. 21, 1188. Maniscalco, W.M., et al. 2005. Pediatric Res. 58, 594. Roper, J.M., et al. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045. Other Applications Babbar, N. and Casero, Jr., R.A. 2006. Cancer Res. 66, 11125. Osella, D., et al. 2005. Inorganica Chimica Acta 358, 1993. Riemschneider, S., et al. 2002. Acta Derm. Venereol. 82, 325.
          生物信息
          化验时间 4 h
          样品类型 Cells
          理化信息
          尺寸
          材料信息
          毒性学信息
          GHS安全信息
          安全信息
          产品使用声明
          预定用途 The Calbiochem® OxyDNA Kit is an in vitro fluorescent protein binding method for the detection of oxidative damage to DNA in fixed permeabilized cells using flow cytometry or fluorescence microscopy. Material of both human and animal origin can be tested.
          储存和货运信息
          货运代码 Ambient Temperature Only
          毒性 Multiple Toxicity Values, refer to MSDS
          储存 +2°C to +8°C
          存储条件 Upon arrival store the entire contents of the kit at 4°C. Note: FITC conjugate must be stored in the dark.
          无需冷冻 No
          包装信息
          运输信息
          补充信息
          试剂盒包含 Wash Concentrate, Binding Protein-FITC Conjugate, and a user protocol.
          规格

          Documentation

          MSDS

          语言
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          分析证书

          标题批号
          500095

          参考

          参考信息概述
          Silva, P.F.N. et al. 2007. Theriogenology 67, 609.
          Babbar, N. and Casero, Jr., R.A. 2006. Cancer Res. 66, 11125.
          Lai, K.N., et al. 2006. Nephrol. Dial. Transplant. 21, 1188.
          Sampson, M.J. et al. 2006. Diabetes Care 29, 283.
          Maniscalco, W.M., et al. 2005. Pediatric Res. 58, 594.
          Osella, D., et al. 2005. Inorganica Chimica Acta 358, 1993.
          Adamek B et al. 2004. Polish Journal of Environmental Studies 13 Suppl. II, 29.
          Roper, J.M., et al. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045.
          Ogawa, Y. et al. 2003. International Journal of Molecular Medicine 11, 27.
          Ogawa, Y. et al. 2003. International Journal of Molecular Medicine 11, 149.
          Sparrow J.R. et al. 2003. Investigative Ophthalmology and Visual Science (14)5, 2245.
          Tao Peng, H-M. et al. 2003. World J Gastroenterol 9, 2186.
          Riemschneider, S., et al. 2002. Acta Derm. Venereol. 82, 325.
          de Zwart, L.L., et al. 1999. Free Radic. Biol. Med. 26, 202.
          Tsurudome, Y., et al. 1999. Carcinogenesis 20, 1573.
          Inoue, M., et al. 1998. Jpn. J. Cancer Res. 89, 691.
          Kasai, H. 1997. Mutat. Res. 387, 147.
          Cooke, C., et al. 1996. Toxicol. Ecotoxicol. News 3, 101.



          Selected Citations
          Roper, J. M., et al. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045.
          Babbar, N., and Casero, R. A., Jr., 2006. Cancer Res. 66, 23.


          用户协议

          版本 23-September-2014 JSW
          形式 50 Tests
          检测方法 Flow cytometry or fluorescence microscopy
          储存 Upon arrival store the entire contents of the kit at 4°C. Note: FITC conjugate must be stored in the dark.
          预定用途 The Calbiochem® OxyDNA Kit is an in vitro fluorescent protein binding method for the detection of oxidative damage to DNA in fixed permeabilized cells using flow cytometry or fluorescence microscopy. Material of both human and animal origin can be tested.
          背景 Oxidative injury to macromolecules is indicated in a wide range of pathological conditions. Damage is mediated via free radicals that can be created by a range of agents, e.g. xenobiotics, environmental toxins, smoking, radiation, ischaemia-reperfusion injury oxidising agents and normal or disturbed metabolic activity. These free radicals may react with DNA causing reversible and irreversible damage. This can lead to mutation, carcinogenesis, teratogenesis or cell death. The probe in the OxyDNA kit is specific for 8-oxoguanine. 8-oxoguanine (as part of the oxidized nucleotide 8-oxyguanosine) is formed during free radical damage to DNA and is a sensitive and specific indicator of oxidative DNA damage. 8-oxoguanine is a particularly important biomarker of oxidative DNA damage as it is formed in relatively large quantities and 8-oxoguanine formation can lead to mutations, such as the substitution of thymine for guanine and cytosine for adenine. Previously, 8-oxoguanine was difficult to detect, requiring HPLC analysis, however by utilizing a binding protein with high avidity and specificity for 8-oxoguanine, the OxyDNA kit provides a simple, convenient, sensitive fluorescence method for detecting for oxidative DNA damage.
          化验原理 The Calbiochem® OxyDNA Kit utilizes a direct fluorescent protein binding technique. After cells have been fixed and permeabilized, the FITC-Conjugate is added and binds to the 8-oxoguanine moiety present in the 8-oxoguanosine of oxidized DNA. The presence of oxidized DNA is indicated by a green/yellow fluorescence that can be read using flow cytometry or fluorescence microscopy.
          提供的材料 • Wash Concentrate (Kit component No. KP10401-55ML): 1 bottle, 55 ml, 25X Tris-buffered Saline/TWEEN® 20 Detergent (TBST), containing ProClin950 concentrate
          • FITC-Conjugate Concentrate (Kit Component No. KP10402-500UL): 1 vial, 0.5 ml Binding Protein conjugated to Fluorescein, containing ProClin950 and Bronidox L.
          要求但未提供的材料 Micropipettes: 20 µl to 100 µl, 200 µl to 1000 µl
          Test tubes
          1 L beaker
          Graduated cylinder
          Reagents for fixing and permeabilising cells
          Deionized/Distilled water
          37°C water bath
          Flow cytometer with appropriate filter combination for FITC (excitation filter 495 nm, barrier filter 515 nm)
          注意事项和建议 The OxyDNA kit contains a chemical known to the State of California to cause birth defects or other reproductive harm (California Prop 65: thimerosal).
          Dispose of all specimens in accordance with good laboratory practice.
          Wear protective clothing, disposable latex gloves and eye protection while handling specimens and performing the assay. Wash hands thoroughly when finished.
          Do not pipette materials by mouth and never eat or drink at the laboratory workbench.
          Do not mix or substitute reagents from different kit lot numbers.
          Care must be taken not to contaminate components and always use fresh pipette tips for each sample and component.
          Do not use reagents that are cloudy or that have precipitated out of solution. High quality distilled or deionised water is required for the Wash Solution. The use of poor quality or contaminated water may lead to background colour in the assay.
          Allow all reagents to come to room temperature (20-25°C) and mix well prior to use.
          Avoid leaving reagents in direct sunlight and/or above 4°C for extended periods.
          Always use clean, preferably disposable, glassware for all reagent preparation.
          制备 Live cells should be shielded from high oxygen tensions (e.g. room air) and unnecessary mechanical stress (e.g. mixing and washing) as these can lead to increased oxidative DNA damage and high levels of fluorescence in untreated/control samples. DNA is more stable in fixed cells.
          试剂制备 • Wash Solution: Dilute the Wash Concentrate 1:25 with diH2O. For example, to prepare 250 ml Wash Solution add 10 ml of Wash Concentrate to 240 ml diH2O. Prepare only the volume of Wash Solution required for the assay. • 1X FITC-Conjugate: Dilute the FITC-Conjugate 1:10 with Wash Solution. For example, to prepare 1 ml 1X FITC-Conjugate add 100 µl FITC-Conjugate to 900 µl Wash Solution. To dispense conjugate, remove cap and pipette directly from the bottle. Keep FITC-Conjugate solutions in the dark when not in use. These dilutions of FITC-Conjugate have been used successfully for a number of cell lines e.g. Hep G2, IMR-32 and CHO, sperm and lymphocytes. In some experimental situations, it may be necessary to optimize the dilution of the conjugate used. This will be dependent on the particular cell type used and the experimental conditions used to induce oxidative damage. If the incorrect concentration of conjugate is used, high background staining and non-specific binding may occur.
          详细的协议 Assay Protocol for Flow Cytometry Assay

          Oxidative DNA damage can be found in most cell types and can be caused by many different mechanisms. As a result, the protocol outlined below should only be regarded as a guideline and the procedures may need to be modified according to the experimental model or cell type being studied.

          1. Fix and permeabilize the cells to be tested.
          2. Wash with Wash Solution.
          3. Add 100 µl 1X FITC-Conjugate to the cell pellet and incubate in the dark for 60 min at room temperature.
          4. Wash with Wash Solution.
          5. Read fluorescence using a flow cytometer at an excitation wavelength of 495 nm and barrier filter of 515 nm.
          特异性 The Calbiochem® OxyDNA Kit is specific for the 8-oxoguanine moiety of 8-oxoguanosine present in oxidized DNA and shows no measurable cross reactivity with unoxidized guanine / guanosine or other unoxidized nucleotides.
          应用参考 Fluorescent Microscopy Lai, K.N., et al. 2006. Nephrol. Dial. Transplant. 21, 1188. Maniscalco, W.M., et al. 2005. Pediatric Res. 58, 594. Roper, J.M., et al. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045. Other Applications Babbar, N. and Casero, Jr., R.A. 2006. Cancer Res. 66, 11125. Osella, D., et al. 2005. Inorganica Chimica Acta 358, 1993. Riemschneider, S., et al. 2002. Acta Derm. Venereol. 82, 325.
          注册商标 Calbiochem® is a registered trademark of EMD Chemicals, Inc.
          Tween® is a registered trademark of ICI Americas, Inc.
          Interactive Pathways™ is a trademark of EMD Chemicals, Inc.

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