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1 使用说明 核糖核酸酶抑制剂适用的pH范围较广但需要至少1mM的DTT存在来维持 活性.使用前请混匀以免浓度不均匀.

2 标准应用 核糖核酸酶抑制剂能应用于体外转录和翻译系统,下面将做描述.若 想了解更多关于体外转录系统的信息,请索取体外转录系统技术文件 #TM016. A 体外转录(未标记的RNA) 下述标准体外转录反应中Rnasin的最终浓度为1u/ul. 经适当调整后,该 反应可用于多种关于体外转录的实验应用中. 5X transcription buffer* 20μl DTT, 100mM 10μl RNasin? Ribonuclease Inhibitor 100u ATP, GTP, CTP and UTP, 2.5mM each** 20μl linearized plasmid DNA, 2C5μg in H2O or TE buffer 2μl RNA polymerase;

SP6, T3 or T7 0C50u nuclease-free water to a final volume of 100μl * 5X transcription buffer:200mM Tris-HCl (pH 7.5)、30mM MgCl

2、10mM spermidine、50mM NaCl. **由4种10mM rNTP储存液等体积配制. 37C40°C 保温 60C120 分钟. B 体外转录(32P-标记的RNA探针) 5X transcription buffer 4μl DTT, 100mM 2μl RNasin? Ribonuclease Inhibitor 20u ATP, GTP and UTP, 2.5mM each 4μl CTP, 100μM 2.4μl linearized template DNA, 0.2C1.0mg/ml in H2O or TE buffer 1μl [-32P]CTP, 50μCi at 10mCi/ml 5μl RNA polymerase, SP6, T3 or T7 1μl nuclease-free water to a final volume of 20μl 37C40°C 保温60分钟,将1体积水和各1体积的10mM ATP,GTP和UTP储 存液混合配制而成. C 体外翻译 在体外翻译系统中加入Rnasin将有效的保证RNA底物不被降解. 示例1:用兔网织红细胞裂解物系统进行体外翻译 Rabbit Reticulocyte Lysate 35μl nuclease-free water 7μl RNasin? Ribonuclease Inhibitor 40u Amino Acid Mixture Minus Methionine, 1mM 1μl [35S]methionine, (1,200Ci/mmol) at 10mCi/ml 4μl RNA template in H2O 2μg Final volume of 50μl 30°C 保温60分钟. 示例2:用TNT? 网织红细胞裂解物或麦芽提取物系统进行体外转录和 体外翻译的联合实验 TNT? Rabbit Reticulocyte Lysate or Wheat Germ Extract 25μl TNT? Reaction Buffer 2μl TNT? T3, T7 or SP6 RNA Polymerase 1μl Amino Acid Mixture Minus Methionine, 1mM 1μl [35S]methionine (1,000Ci/mmol) at 10mCi/ml 4μl RNasin? Ribonuclease Inhibitor, 40u/μl 40u DNA template 1μg Nuclease-free water to a final volume of 50μl 30°C保温 60C120 分钟. 1. Blackburn, P. and Moore, S. (1982) In: The Enzymes, Vol. XV, Part B, Academic Press,New York. 2. Blackburn, P., Wilson, G. and Moore, S. (1977) Ribonuclease inhibitor from human placenta. Purification and properties. J. Biol. Chem. 252, 5904C 10. 3. Lee, F.S., Auld, D.S. and Vallee, B.L. (1989) Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin. Biochemistry 28, 219C24. 4. Lee, F.S., Shapiro, R. and Vallee, B.L. (1989) Tight-binding inhibition of angiogenin and ribonuclease A by placental ribonuclease inhibitor. Biochemistry 28, 225C30. 5. Shapiro, R. and Vallee, B.L. (1987) Human placental ribonuclease inhibitor abolishes both angiogenic and ribonucleolytic activities of angiogenin. Proc. Natl. Acad. Sci.USA 84, 2238C41. 6. Polakowski, I.J. et al. (1993) A ribonuclease inhibitor expresses anti-angiogenic properties and leads to reduced tumor growth in mice. Am. J. Pathol. 143, 507C17. ??参考文献? ??建议使用方法?