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中性pH键断裂剂TCEP溶液使用说明

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  • 作者:昊帆研发部
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  • 发布时间:2021-05-13 13:28
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【概要描述】TCEP溶液作为中性键断裂剂,是一种有效的、通用的、无臭的且不含硫醇的还原剂,广泛应用于蛋白质和其他涉及二硫键还原的研究。

中性pH键断裂剂TCEP溶液使用说明

【概要描述】TCEP溶液作为中性键断裂剂,是一种有效的、通用的、无臭的且不含硫醇的还原剂,广泛应用于蛋白质和其他涉及二硫键还原的研究。

  • 分类:新闻中心
  • 作者:昊帆研发部
  • 来源:
  • 发布时间:2021-05-13 13:28
  • 访问量:
详情
中性pH键断裂剂TCEP溶液使用说明
 
产品描述

键断裂剂TCEP溶液,中性pH,5mL,为中性的和稳定的0.5M TCEP溶液。

 
储存方法

建议室温保存,未使用时保持密闭。

 
使用说明

TCEP溶液作为中性键断裂剂,是一种有效的、通用的、无臭的且不含硫醇的还原剂,广泛应用于蛋白质和其他涉及二硫键还原的研究(图1)。该产品也是SDS-PAGE样品缓冲液中β-巯基乙醇或DTT的高效替代品,其中性pH的特性有助于获得清晰的条带,避免将蛋白质暴露在TCEP•HCl的强酸性环境中,从而避免发生水解和糖基改性。

三烷基膦化合物还原蛋白质二硫键的能力和优点早已为人们所熟知[1][2],但由于在水中溶解度差以及难闻的气味导致应用较少。磷酸盐在水溶液中较稳定,可以选择性断裂二硫键,并且对其他官能团几乎没有作用[2],三(2-羧乙基)膦(TCEP)的发现克服了三烷基膦化合物的缺点从而被广泛应用[3]~[25]

图1 TCEP还原有机二硫键

 
聚丙烯酰胺凝胶电泳步骤

1、将中性pH键断裂剂TCEP溶液加到50mM(浓度稀释十倍)的2X SDS样品缓冲液中,用于三甘氨酸凝胶(25mM,20%甘油,4%SDS,0.005%溴酚蓝,pH6.8)的还原;

2、将等体积的样品和2X浓度的还原剂样品缓冲液加到微量离心管中,并于95℃热水浴中保温5分钟;

3、以上样品冷却,快速离心去除不溶性蛋白质聚集体,然后装入凝胶中。

 

参考文献:

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2. Kirley, T.L. (1989). Reduction and fluorescent labeling of cyst(e)ine-containing proteins for subsequent structural analysis. Anal Biochem 180:231. 

3. Burns, J.A., et al. (1991). Selective reduction of disulfides by tris-(2-carboxyethyl)-phosphine. J Org Chem 56:2648-50. 

4. Han, J., et.al. (1999). Tris[2-carboxyethyl]phosphine – A reducing agent with versatile applications including cleavage of disulfide bonds and 

quantitation of numerous oxidants. Previews 2(4):16-21. 

5. Han, J., et al. (1993). Modification of catalytic properties of chicken liver fructose 1,6-bisphosphatase by allicin. Biochem Mol Biol Int 31:1007-15. 

6. Han, J.C. and Han, G.Y. (1994). A procedure for quantitative determination of tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable 

and effective than dithiothreitol. Anal Biochem 220:5-10. 

7. Mery, J., et al. (1993). Disulfide linkage to polyacrylic resin for automated Fmoc peptide synthesis, immunochemical applications of peptide resin and 

mercaptoamide peptide. Int J Pept Protein Res 42:44-52. 

8. Gray, W.R. (1993). Disulfide structures of highly bridged peptides: a new strategy for analysis. Protein Sci 2:1732-48. 

9. Fisher, W.H., et al. (1993). In situ reduction suitable for matrix-assisted laser desorption/ionization and liquid secondary ionization using tris(2- 

carboxyethyl)phosphine. Rapid Commun. Mass Spectrom 7:225-8. 

10. Gozlan, H., et al. (1994). Anoxic LTP is mediated by the redox modulatory site of the NMDA receptor. J Neorophys 72:3017-22. 

11. Gozlan, H., et al. (1995). In CA1 hippocampal neurons, the redox state of NMDA receptors determine LTP expressed by NMDA but not by AMPA 

receptors. J Neurophys 73:2612-17. 

12. Bieri, S., et al. (1995). Disulfide bridges of a cysteine-rich repeat of the LDL receptor ligand-binding domain. Biochemistry 34:13059-65. 

13. Tam, J.P., et al. (1995). Peptide synthesis using unprotected peptides through orthogonal coupling methods. Proc Natl Acad Sci USA 92:12485-9. 

14. Blauenstein, P., et al. (1995). Experience with the iodine-123 and technetium-99m labelled anti-granulocyte antibody MAb47: a comparison of 

labelling methods. Eur J Nucl Med 22:690-8. 

15. Gorman, J.J., et al. (1996). Use of 2,6-dihydroxyacetophenone for analysis of fragile peptides, disulphide bonding and small proteins by matrix

assisted laser desorption/ionization. Rapid Commun Mass Spectrom 10:529-36. 

16. Hirsch, J.C., et al. (1996). Enhanced NMDAR-dependent epileptiform activity is controlled by oxidizing agents in a chronic model of temporal lobe 

epilepsy. J Neurosci 76:4185-9. 

17. Quesada, O., et al. (1996). Redox sites of NMDA receptors can modulate epileptiform activity in hippocampal slices from kainic acid-treated rats. 

Neurosci Lett 212:171-4. 

18. Kirsch, T., et al. (1996). Cloning, high-yield expression in Escherichia coli, and purification of biologically active HIV-1 Tat protein. Protein Express 

Purif 8:75-84. 

19. Haniu, M., et al. (1996). Glial cell line-derived neurotrophic factor: selective reduction of the intermolecular disulfide linkage and characterization of 

its disulfide structure. Biochemistry 35:16799-05. 

20. White, C.E., et al. (1996). The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide 

bonding pattern. Proc Natl Acad Sci 93:10177-82. 

21. Xiao, Y., et al. (1997). Involvement of disulfide bonds in the renal sodium/phosphate co-transporter NaPi-2. Biochem J 323:401-8. 

22. Wu, J. and Watson, J.T. (1997). A novel methodology for assignment of disulfide bond pairings in proteins. Protein Sci 6:391-8. 

23. Bernard, C.L., et al. (1997). Redox modulation of synaptic responses and plasticity in rat CA1 hippocampal neurons. Exp Brain Res 113:343-52. 

24. Riddles, P.W., et al. (1979). Ellman's reagent: 5,5´-dithiobis (2-nitroben-zoic acid) - A reexamination. Anal Biochem 94:75-81. 

25. Cavallito, C.G., et al. (1944). Allicin, the antibacterial principle of Allium salivum. II. Determination of the chemical structure. J Am Chem Soc 66:1952-4.

 

 

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