ATP检测试剂盒
产品编号 S0026 产品名称 ATP检测试剂盒(ATP Assay Kit) 包装 >200次 产品简介:
ATP检测试剂盒(ATP Assay Kit)可以用于检测普通溶液中或细胞或组织内的ATP(adenosine 5’-triphosphate)水平。ATP,作为最重要的能量分子在细胞的各种生理、病理过程中起着重要作用。ATP水平的改变,会影响许多细胞的功能。通常细胞在凋亡、坏死或处于一些毒性状态下,ATP水平会下降,而高葡萄糖刺激等对于一些细胞可以上调细胞内ATP水平。通常ATP水平的下降表明线粒体的功能受损或下降,在细胞凋亡时ATP水平的下降通常和线粒体的膜电位下降同时发生。
本试剂盒根据萤火虫荧光素酶(firefly luciferase)化荧光素产生荧光时需要ATP提供能量研制而成。当萤火虫荧光素酶和荧光素都过量时,在一定的浓度范围内荧光的产生和ATP的浓度成正比。这样就可以高灵敏地检测溶液中的ATP浓度。本检测试剂盒可以检测浓度低达0.1nmol/L的ATP。而常规的细胞或组织裂解液中ATP的浓度仅为0.1-1μmol/L,一些常见细胞的细胞内ATP水平约为10 nmol/mg蛋白。
检测细胞或组织内ATP浓度时,用本试剂盒提供的裂解液裂解后即可以测定ATP浓度。 本试剂盒方便快捷,通常10-20个样品可以在30-60分钟内测定完毕。 本试剂盒至少可以检测200个样品。
包装清单: 产品编号 S0026-1 S0026-2 S0026-3 S0026-4 — 产品名称 ATP检测试剂 ATP检测试剂稀释液 ATP标准溶液 (0.5mM) ATP检测裂解液 说明书 包装 0.2 ml(50μl/管,共4管) 20ml 0.1ml 100ml 1份 保存条件:
-20℃保存,半年有效。-70℃保存,一年有效。ATP检测试剂需避光保存。
注意事项:
ATP检测试剂中含有荧光素酶,反复冻融会导致其逐渐失活。为取得较好的使用效果,冻融的次数不宜超过3次。ATP检测试剂稀释成ATP检测工作液后,最好一次用完,不宜冻存后再使用。
ATP,特别是裂解后样品中的ATP在室温不太稳定,需在4℃或冰上操作。ATP在冰上可以稳定长达6小时。
本试剂盒需使用luminometer(检测荧光素酶报告基因时所用的仪器)。如果没有luminometer,也可以使用液闪仪。液
闪仪的测定效果取决于液闪仪的检测灵敏度和检测精度。 为了您的安全和健康,请穿实验服并戴一次性手套操作。
使用说明:
1. 样品测定的准备:(注意:样品裂解需在4℃或冰上操作) 对于贴壁细胞:
吸除培养液,按照6孔板每孔加入200微升裂解液的比例(即相当于细胞培养液量2毫升的1/10)加入裂解液,裂解细胞。裂解细胞时为了裂解充分,可以使用移液器进行反复吹打或晃动培养板使裂解液充分接触并裂解细胞。通常细胞在接触裂解液后会立即裂解。裂解后4℃ 12000g离心5-10分钟,取上清,用于后续的测定。 对于悬浮细胞:
用离心管离心沉淀细胞,弃上清,轻轻弹散细胞,按照6孔板每孔的细胞量加入200微升裂解液的比例加入裂解液,裂解细胞。裂解细胞时为了裂解充分可以弹击离心管管底或适当Vortex使裂解液充分接触并裂解细胞。通常细胞在接触裂解液后会立即裂解。裂解后4℃ 12000g离心5-10分钟,取上清,用于后续的测定。 对于组织样品:
按照每20毫克组织加入约100-200微升裂解液的比例加入裂解液,然后用玻璃匀浆器或其它匀浆器进行匀浆。充分匀浆可以确保组织被完全裂解。裂解后4℃ 12000g离心5-10分钟,取上清,用于后续的测定。 2. 标准曲线测定的准备:
冰浴上溶解待用试剂,把ATP标准溶液用ATP检测裂解液稀释成适当的浓度梯度。具体的浓度需根据样品中ATP的浓度而
定。初次检测可以检测0.1、1和10微摩尔/升这几个浓度,在后续的实验中根据样品中ATP的浓度对标准品的浓度进行适当调节。
3. ATP检测工作液的配制:
按照每个样品或标准品需100微升ATP检测工作液的比例配制适当量的ATP检测工作液。把待用试剂在冰浴上溶解。取适当量的ATP检测试剂,按照1:100的比例用ATP检测试剂稀释液稀释ATP检测试剂。例如50微升ATP检测试剂可以加入5毫升ATP检测试剂稀释液配制成5毫升ATP检测工作液。稀释后的ATP检测试剂即为用于后续实验的ATP检测工作液。ATP检测工作液可在冰浴上暂时保存。 4. ATP浓度的测定:
(1) 加100微升ATP检测工作液到检测孔或检测管内。室温放置3-5分钟,以使本底性的ATP全部被消耗掉,从而降低
本底。可以一次性把10-20个检测孔或检测管分别加上100微升ATP检测工作液,从而节省时间。
(2) 在检测孔或检测管内加上10-100微升样品或标准品,迅速用枪(微量移液器)混匀,至少间隔2秒后,立即用
luminometer或液闪仪测定RLU值或CPM。(如果样品中的ATP浓度比较低则可以加100微升样品,如果样品中ATP浓度比较高则可以加较小体积的样品,同时标准品的也需要使用相同的体积。如果样品中ATP的浓度特别高,可以用MilliQ级的纯水或重蒸水稀释样品后再测定,这样ATP标准液也要作类似于样品的稀释,保证检测时标准品中ATP的含量和样品基本一致。本试剂盒在加100微升样品时,大致在0.1nmol/L-100nmol/L的浓度范围内有很好的线性关系。)
(3) 根据标准曲线计算出样品中ATP的浓度。
(4) 为了消除样品制备时由于蛋白量的差异而造成的误差,可以用碧云天生产的BCA蛋白浓度测定试剂盒
(P0009/P0010/P0010S/P0011/P0012/P0012S)测定样品中的蛋白浓度。然后把ATP的浓度换算成nmol/mg蛋白的形式。
常见问题:
1.
Luminometer和荧光分光光度计有何不同?
荧光分光光度计检测的样品本身不能发光,样品需要由特定波长的激发光激发,然后才能产生荧光并被荧光分光光度计检测。Luminometer检测的样品本身可以发光,不需要激发光进行激发。也就是说Luminometer是检测化学发光的仪器。有些型号的荧光分光光度计也具有luminometer的功能,即也可以检测化学发光。您所使用的荧光分光光度计能否用于化学发光的测定请仔细阅读该仪器的说明书。
可以进行荧光素酶报告基因检测的仪器是否就可以用于本试剂盒的ATP检测?
是。荧光素酶报告基因的检测原理和本ATP检测试剂盒的原理相同,可以用相同的仪器测定,并且可以选择相同的测定参数,例如检测前等待时间为2秒,检测时间为10秒等。
2.
使用本产品的文献:
1. Zhu CH, Lu FP, He YN, Han ZL, Du LX.
Regulation of avilamycin biosynthesis in Streptomyces viridochromogenes: effects of glucose, ammonium ion, and inorganic phosphate. Appl Microbiol Biotechnol. 2007 Jan;73(5):1031-8. 2. Wang YM, Pu P, Le WD.
ATP depletion is the major cause of MPP+ induced dopamine neuronal death and worm lethality in alpha-synuclein transgenic C. elegans. Neurosci Bull. 2007 Nov;23(6):329-35. 3. Peng X, Li F, Li S, Zhu Y.
Expression of a mitochondrial gene orfH79 from the CMS-HongLian rice inhibits Saccharomyces cerevisiae growth and causes excessive ROS accumulation and decrease in ATP.
Biotechnol Lett. 2009 Mar;31(3):409-14. Epub 2008 Nov 28.
4. Sheng B, Gong K, Niu Y, Liu L, Yan Y, Lu G, Zhang L, Hu M, Zhao N, Zhang X, Tang P, Gong Y.
Inhibition of gamma-secretase activity reduces Abeta production, reduces oxidative stress, increases mitochondrial activity and leads to reduced vulnerability to apoptosis: Implications for the treatment of Alzheimer's disease. Free Radic Biol Med. 2009 May 15;46(10):1362-75.
5. Chen K, Zhang Q, Wang J, Liu F, Mi M, Xu H, Chen F, Zeng K.
Taurine protects transformed rat retinal ganglion cells from hypoxia-induced apoptosis by preventing mitochondrial dysfunction. Brain Res. 2009 Jul 7;1279:131-8. Epub 2009 May 7.
6. Chu L, Wang J, Wang B, Xing XH, Yan S, Sun X, Jurcik B.
Changes in biomass activity and characteristics of activated sludge exposed to low ozone dose. Chemosphere. 2009 Sep;77(2):269-72. 7. Sun Q, Hu C, Hu J, Li S, Zhu Y.
Quantitative proteomic analysis of CMS-related changes in Honglian CMS rice anther. Protein J. 2009 Oct;28(7-8):341-8.
8. Wang H, Chen XX, Wang LR, Mao YD, Zhou ZM, Sha JH. AF-2364 is a prospective spermicide candidate.
Asian J Androl. 2010;12(3):322-35. Epub 2010 Apr 26. 9. Cao X, Zhang Y, Zou L, Xiao H, Chu Y, Chu X.
Persistent oxygen-glucose deprivation induces astrocytic death through two different pathways and calpain-mediated proteolysis of cytoskeletal proteins during astrocytic oncosis.
Neurosci Lett. 2010;479(2):118-22. Epub 2010 May 21.
10. Liu Q, Zhang Y, Lin Z, Shen H, Chen L, Hu L, Jiang H, Shen X.
Danshen extract 15,16-dihydrotanshinone I functions as a potential modulator against metabolic syndrome through multi-target pathways. J Steroid Biochem Mol Biol. 2010;120(4-5):155-63. Epub 2010 Apr 7. 11. Li SF, Liu HX, Zhang YB, Yan YC, Li YP.
The protective effects of alpha-ketoacids against oxidative stress on rat spermatozoa in vitro. Asian J Androl. 2010;12(2):247-56. Epub 2009 Dec 14.
12.Yang Q, Gong ZJ, Zhou Y, Yuan JQ, Cheng J, Tian L, Li S, Lin XD, Xu R, Zhu ZR, Mao C. Role of Drosophila alkaline ceramidase (Dacer) in Drosophila development and longevity. Cell Mol Life Sci. 2010 May;67(9):1477-90.
13.Zhang W, Shi Y, Chen Y, Yu S, Hao J, Luo J, Sha X, Fang X.
Enhanced antitumor efficacy by paclitaxel-loaded pluronic P123/F127 mixed micelles against non-small cell lung cancer based on passive tumor targeting and modulation of drug resistance.
Eur J Pharm Biopharm. 2010 Aug;75(3):341-53. 14.Gong HS, Meng XC, Wang H
Mode of action of plantaricin MG, a bacteriocin active against Salmonella typhimurium. J Basic Microbiol. 2010 Dec;50 Suppl 1:S37-45.
15.Zhu SS, Ren Y, Zhang M, Cao JQ, Yang Q, Li XY, Bai H, Jiang L, Jiang Q, He ZG, Chen Q.
Wld(S) protects against peripheral neuropathy and retinopathy in an experimental model ofdiabetes in mice. Diabetologia. 2011 Sep;54(9):2440-50.
16.Zhang W, Shi Y, Chen Y, Ye J, Sha X, Fang X.
Multifunctional Pluronic P123/F127 mixed polymeric micelles loaded with paclitaxel for thetreatment of multidrug resistant tumors. Biomaterials. 2011 Apr;32(11):2894-906.
17.Cui J, Ou S, He WJ, Du L, Zhao YD, Ruan HZ.
Prevention of extracellular ADP-induced ATP accumulation of the cultured rat spinal astrocytes viaP2Y(1)-mediated inhibition of AMPK. Neurosci Lett. 2011 Oct 10;503(3):244-9.
18.Cao X, Yang M, Wei RC, Zeng Y, Gu JF, Huang WD, Yang DQ, Li HL, Ding M, Wei N, Zhang KJ, Xu B, Liu XR, Qian QJ, Liu XY. Cancer targeting Gene-Viro-Therapy of liver carcinoma by dual-regulated oncolytic adenovirusarmed with TRAIL gene. Gene Ther. 2011 Aug;18(8):765-77.
19.Chang WQ, Wu XZ, Cheng AX, Zhang L, Ji M, Lou HX.
Retigeric acid B exerts antifungal effect through enhanced reactive oxygen species anddecreased cAMP. Biochim Biophys Acta. 2011 May;1810(5):569-76. 20.Cui Y, Zhao Y, Tian Y, Zhang W, Lü X, Jiang X.
The molecular mechanism of action of bactericidal gold nanoparticles on Escherichia coli. Biomaterials. 2012 Mar;33(7):2327-33.
21.Lin LF, Xue XY, Liao MJ, Xiao F, Lv RH, Luo HM.
Neurotrophic effects of magnesium fructose 1, 6-diphosphate on cortical neurons. Int J Neurosci. 2012 May;122(5):248-54. Epub 2012 Jan 20. 22.Feng Q, Yu AF, Chu LB, Chen HZ, Xing XH
Mechanistic Study of On-site sludge Reduction in a Baffled Bioreactor Consisting of three series of Alternating Aerobic and Anaerobic Compartments Biochemical Engineering Journal,2012 Aug,67(15),45-51
23.Chen Y, Yin Q, Ji X, Zhang S, Chen H, Zheng Y, Sun Y, Qu H, Wang Z, Li Y, Wang X, Zhang K, Zhang L, Shi J.
Manganese oxide-based multifunctionalized mesoporous silica nanoparticles for pH-responsiveMRI, ultrasonography and circumvention of MDR in cancer cells.
Biomaterials. 2012 Oct;33(29):7126-37.
24.Peng Y, Dong D, Jiang C, Yu B, Wang X, Ji Y.
Relationship between respiration deficiency and azole resistance in clinical Candida glabrata. FEMS Yeast Res. 2012 Jun 19.
25.Yu SJ, Liu HC, Ling-Ling E, Wang DS, Zhu GX
Proliferation and differentiation of osteoblasts from the mandible of osteoporotic rats. Exp Biol Med (Maywood). 2012 Apr;237(4):395-406 26.Liu D, Liu S, Jing X, Li X, Li W, Huang Y
Necrosis of cervical carcinoma by dichloroacetate released from electrospun polylactide mats. Biomaterials. 2012 Jun;33(17):4362-9.
27.Li XY, Jing CQ, Zang XY, Yang S, Wang JJ.
Toxic cytological alteration and mitochondrial dysfunction in PC12 cells induced by 1-octyl-3-methylimidazolium chloride. Toxicol In Vitro. 2012 Jul 23.
28.Yuan Y, Peng L, Gong-Hua H, Lu D, Xia-Li Z, Yu Z, Cai-Gao Z.
Chi-square analysis of the reduction of ATP levels in L-02 hepatocytes by hexavalent chromium. Braz J Med Biol Res. 2012 Jun;45(6):482-7.
29.Song GX, Shen YH, Liu YQ, Sun W, Miao LP, Zhou LJ, Liu HL, Yang R, Kong XQ, Cao KJ, Qian LM, Sheng YH Overexpression of FABP3 promotes apoptosis through inducing mitochondrial impairment inembryonic cancer cells. J Cell Biochem. 2012 Jul 2.
30.Zhu S, Wang Y, Chen M, Jin J, Qiu Y, Huang M, Huang Z
Protective Effect of Schisandrin B Against Cyclosporine A-Induced Nephrotoxicity In Vitro and InVivo. Am J Chin Med. 2012 2012;40(3):551-566.
31.Yu Ping Li, Hu Su, Xiao Fang Pi, Yan Chun Gong, Xiang Yuan Xiong, Guang Jie Wu, Zi Ling Li
The Stimulatory Activities of Baicalein and Baicalin Compounds Derived from Scutellaria baicalensis on Insulin Secretion In Vitro Advanced Materials Research,2012, 554-556, 1673
32.Huang HY, Cai JF, Liu QC, Hu GH, Xia B, Mao JY, Wu DS, Liu JJ, Zhuang ZX.
Role of poly(ADP-ribose) glycohydrolase in the regulation of cell fate in response tobenzo(a)pyrene. Exp Cell Res. 2012 Mar 10;318(5):682-90.
33.Yuan Y, Ming Z, Gong-Hua H, Lan G, Lu D, Peng L, Feng J, Cai-Gao Z.
Cr(VI) induces the decrease of ATP level and the increase of apoptosis rate mediated by ROS orVDAC1 in L-02 hepatocytes. Environ Toxicol Pharmacol. 2012 Jul 7;34(2):579-587. [Epub ahead of print] 34.Yang Y,Li P, Hu GH, Zhou Y, Dai L, Zhong CG
Association between VDAC1 mRNA expression and intracellular ATP levels of cultured L-02 hepatocytes during hexavalent chromium toxicity African Journal of Biotechnology Vol. 11 (2), pp. 264-272, 5 January, 2012
因篇幅问题不能全部显示,请点此查看更多更全内容