Differential in vivo Thiol Trapping with N-ethylmaleimide (NEM) and 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS)
使用n-乙基马来酰亚胺（NEM）和4-乙酰氨基-4'-马来酰亚胺-2,2'-二磺酸（AMS）进行体内硫醇差速俘获
发布: 2012年10月20日第2卷第20期 DOI: 10.21769/BioProtoc.272 浏览次数: 14360
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参见作者原研究论文
The authors used this protocol in:
Mar 2012
Abstract
This protocol is used to compare the in vivo redox status of Escherichia coli and Vibrio cholerae protein before and after HOCl treatment. For example, I examined whether the EF-Tu protein is reduced or oxidized in the referenced publication. This protocol should work for other proteins and other oxidative stress treatments. You will need the antibody for your protein to visualize your protein on western blot.
Keywords: Thiol trap (硫醇陷阱)
Oxidation (氧化)
Redox status (氧化还原状态)
Materials and Reagents
- Bacteria (this protocol works for E. coli MG1655 and Vibrio cholerae O395 strains, I didn’t try other bacteria)
- HOCl (Sigma-Aldrich)
- Trichloroacetic acid (TCA)
- Urea
- Tris-HCl
- EDTA
- SDS
- N-ethylmaleimide (NEM)
- DTT
- 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS)
- Polyclonal antibodies against your protein and appropriate secondary antibodies
- LB media (see Recipes)
- DAB buffer (see Recipes)
Equipment
- Centrifuges
- Western blotting equipment
- Spectrometer
Procedure
文章信息
版权信息
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Wholey, W. W. (2012). Differential in vivo Thiol Trapping with N-ethylmaleimide (NEM) and 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS). Bio-protocol 2(20): e272. DOI: 10.21769/BioProtoc.272.
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分类
微生物学 > 微生物生物化学 > 其它化合物
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