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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   

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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

  1. Bacteria (this protocol works for E. coli MG1655 and Vibrio cholerae O395 strains, I didn’t try other bacteria)
  2. HOCl (Sigma-Aldrich)
  3. Trichloroacetic acid (TCA)
  4. Urea
  5. Tris-HCl
  6. EDTA
  7. SDS
  8. N-ethylmaleimide (NEM)
  9. DTT
  10. 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS)
  11. Polyclonal antibodies against your protein and appropriate secondary antibodies
  12. LB media (see Recipes)
  13. DAB buffer (see Recipes)

Equipment

  1. Centrifuges
  2. Western blotting equipment
  3. Spectrometer

Procedure

  1. Cultivate bacterial strains in LB media at 37 °C, shack 250 rpm, until OD600 of 0.4-0.5 was reached.
  2. Collect 1 ml cells as untreated control, 2 tubes of 1 ml cells as labeling controls (see step 13 for detail) and then add 3 mM HOCl (final concentration) to rest of culture directly and continue incubation at 37 °C, shack 250 rpm.
  3. After the 20 min of HOCl stress treatment, collect 1 ml of treated cells.
  4. Acidify collected samples and precipitate proteins with TCA to a final concentration of 10%. This step also prevents any further thiol oxidation.
  5. Incubate samples 30 min on ice, pellet precipitated proteins by centrifugation [13,000 rpm (15.7 x g), 20 min, 4 °C].
  6. Rinse protein pellet (not resuspend) with 100 μl 5% TCA, remove TCA with centrifugation [13,000 rpm (15.7 x g), 2 min, 4 °C]. Carefully remove all TCA, residual acid will interfere subsequent labeling.
  7. Resuspend the protein pellet in 50 μl DAB buffer supplemented with 100 mM NEM to irreversibly alkylate all in vivo reduced cysteines.
  8. Incubate samples for 30 min at 25 °C, with 1,300 rpm shaking.
  9. Precipitate the proteins again with TCA to remove any unbound NEM, and pellet by centrifugation.
  10. Resuspend protein pellets in 50 μl DAB buffer supplemented with 10 mM DTT to reduce all in vivo oxidized cysteines, and incubate for 1 h at 25 °C, with 1,300 rpm shaking.
  11. Remove excess DTT by TCA precipitation and centrifugation.
  12. For differential thiol trapping with AMS, modify all newly accessible cysteines (previously oxidized) with 10 mM of the thiol-specific alkylation reagent AMS, which adds 500 Da mass to every modified cysteine.
  13. As controls, we also prepared fully NEM-labeled proteins (represent the fully reduced species) and fully AMS-labeled proteins (represent the fully oxidized protein species).
    1. Take untreated cell aliquots, precipitate with TCA and resuspend in 50 μl DAB buffer supplemented with 10 mM DTT to reduce all in vivo thiol modifications.
    2. After incubation of the samples for 1 h at 25 °C, precipitate proteins with TCA to remove DTT, centrifuge and resuspend in 50 μl DAB buffer supplemented with either 100 mM NEM or 10 mM AMS.
  14. Separate proteins on SDS-PAGE and visualize your protein by western blot analysis mass addition significantly slows the migration of AMS-labeled proteins and allows direct visualization of the in vivo redox status of proteins.

Recipes

  1. LB media
  2. DAB buffer
    6 M Urea
    200 mM Tris-HCl (pH 8.5)
    10 mM EDTA
    0.5% w/v SDS

Acknowledgments

The protocol described here is adapted from one reported previously (Wholey et al., 2012).

References

  1. Wholey, W. Y. and Jakob, U. (2012). Hsp33 confers bleach resistance by protecting elongation factor Tu against oxidative degradation in Vibrio cholerae. Mol Microbiol 83(5): 981-991.

简介

该方案用于比较HOCl处理之前和之后大肠杆菌和霍乱弧菌蛋白的体内氧化还原状态。 例如,我在所引用的出版物中检查了EF-Tu蛋白是否被还原或氧化。 这个协议应该适用于其他蛋白质和其他氧化应激治疗。 您将需要您的蛋白质的抗体在蛋白质印迹上可视化您的蛋白质。

关键字:硫醇陷阱, 氧化, 氧化还原状态

材料和试剂

  1. 细菌(此协议适用于大肠杆菌MG1655和霍乱弧菌O395菌株,我没有尝试其他细菌)
  2. HOCl(Sigma-Aldrich)
  3. 三氯乙酸(TCA)
  4. 尿素
  5. Tris-HCl
  6. EDTA
  7. SDS
  8. N-乙基马来酰亚胺(NEM)
  9. DTT
  10. 4-乙酰氨基-4'-马来酰亚胺基茋-2,2'-二磺酸(AMS)
  11. 针对您的蛋白和适当的二级抗体的多克隆抗体
  12. LB媒体(见配方)
  13. DAB缓冲区(参见配方)

设备

  1. 离心机
  2. Western印迹设备
  3. 光谱仪

程序

  1. 在LB培养基中在37℃下以250rpm的转速培养细菌菌株,直到OD 600达到0.4-0.5。
  2. 收集1ml细胞作为未处理的对照,2管1ml细胞作为标记对照(详见步骤13)(然后向终止培养物中直接加入3mM HOCl(最终浓度),并继续在37℃,小室250rpm 。
  3. 在HOCl应激治疗20分钟后,收集1ml处理的细胞
  4. 酸化收集的样品并用TCA沉淀蛋白质至终浓度为10%。 该步骤还防止任何进一步的硫醇氧化
  5. 在冰上孵育样品30分钟,通过离心[13,000rpm(15.7×g/g),20分钟,4℃]沉淀沉淀的蛋白质。
  6. 用100μl5%TCA冲洗蛋白质沉淀(不再悬浮),用离心[13,000rpm(15.7×g),2分钟,4℃]除去TCA。小心地清除所有TCA,残留的酸会干扰后续标签
  7. 将蛋白质沉淀重悬于补充有100mM NEM的50μlDAB缓冲液中,以不可逆地烷基化所有在体内减少的半胱氨酸。
  8. 孵育样品30分钟,在25°C,1,300 rpm摇动
  9. 用TCA再次沉淀蛋白质以除去任何未结合的NEM,并通过离心沉淀
  10. 将蛋白质沉淀重悬于补充有10mM DTT的50μlDAB缓冲液中,以减少所有体内氧化的半胱氨酸,并在23℃,1,300rpm振荡下孵育1小时。
  11. 通过TCA沉淀和离心除去过量的DTT
  12. 对于使用AMS的差异巯基捕获,用10mM的硫醇特异性烷基化试剂AMS修饰所有新可及的半胱氨酸(先前被氧化),其对每个修饰的半胱氨酸添加500Da质量。
  13. 作为对照,我们还制备了完全NEM标记的蛋白质(代表完全还原的物种)和完全AMS标记的蛋白质(代表完全氧化的蛋白质种类)。
    1. 取未处理的细胞等分试样,用TCA沉淀并重悬于补充有10mM DTT的50μlDAB缓冲液中以减少所有的体内巯基修饰。
    2. 在25℃下孵育样品1小时后,用TCA沉淀蛋白质以除去DTT,离心并重悬于补充有100mM NEM或10mM AMS的50μlDAB缓冲液中。
  14. 在SDS-PAGE上分离蛋白质并通过蛋白质印迹分析显示蛋白质质量添加显着减慢AMS标记的蛋白质的迁移,并允许蛋白质的体内氧化还原状态的直接可视化。

食谱

  1. LB媒体
  2. DAB缓冲区
    6 M尿素
    200mM Tris-HCl(pH8.5) 10 mM EDTA
    0.5%w/v SDS

致谢

此处描述的方案改变自之前报道的方案(Wholey等人,2012)。

参考文献

  1. Wholey,W.Y。和Jakob,U。(2012)。 Hsp33 通过保护延伸因子Tu抗氧化降解而赋予抗漂白性, em> Vibrio cholerae 。 Mol Microbiol 83(5):981-991。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
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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