In vitro Ubiquitin Dimer Formation Assay
体外泛素二聚体形成检测实验   

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参见作者原研究论文

本实验方案简略版
Journal of Experimental Botany
May 2016

Abstract

The process of protein ubiquitination typically consists of three sequential steps to add an ubiquitin (Ub) or Ub chain to a substrate protein, requiring three different enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3). Most E2s possess the classical E2 activity in forming E2-Ub complex through a thioester linkage, in presence of an E1 and Ub. Additionally, some E2s have the ability of catalyzing the formation of free Ub dimer. Such activity indicates an important role of these E2s in ubiquitination pathway. Thus, we developed an in vitro Ub dimer formation assay to determine the activity of certain E2s. Moreover, by using Ub mutants, in which different lysine residues are mutated, the specific linkage of dimer can also be determined.

Keywords: Ubiquitination (泛素化), Ubiquitin dimer formation (泛素二聚体形成), E2 (E2), Arabidopsis UBC22 (拟南芥UBC22), K11 linkage (K11连接)

Background

The existing protocols for E2 conjugation initiation assay (without adding E3 and substrate) aim to detect the thioester linkage (E2-S-Ub). Our method focuses on the E2 activity of catalyzing free Ub dimer formation (Ub-Ub). It provides a convenient way to detect an important biochemical feature of E2 in different species. Further, the specific linkage of dimer can be determined by using different Ub mutants.

Materials and Reagents

  1. 1.5 ml polypropylene tubes
  2. Polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, catalog number: 162-0177 )
  3. AmershamHyperfilmTM ECL (GE Healthcare, catalog number: 28906836 )
  4. Purified human recombinant E1 (BOSTONBIOCHEM, catalog number: K-995 )
  5. Qiagen Ni-NTA Spin Kit (QIAGEN, catalog number: 31314 )
  6. Purified human recombinant Ub (BOSTONBIOCHEM, catalog number: K-995 )
  7. Purified human recombinant Ub with the lysine 11 (K11) residue mutated (Ub-K11R) (BOSTONBIOCHEM, catalog number: UM-K11R )
  8. Purified human recombinant Ub with the lysine 48 (K48) residue mutated (Ub-K48R) (BOSTONBIOCHEM, catalog number: UM-K48R )
  9. Purified human recombinant Ub with the lysine 63 (K63) residue mutated (Ub-K63R) (BOSTONBIOCHEM, catalog number: UM-K63R )
  10. 10x reaction buffer (BOSTONBIOCHEM, catalog number: K-995 )
  11. Mg-ATP solution (BOSTONBIOCHEM, catalog number: K-995 )
  12. 4x non-reducing loading buffer (BOSTONBIOCHEM, catalog number: K-995 )
  13. SDS-PAGE gel
  14. Skimmed milk powder
  15. Anti-ubiquitin antibody (Cell Signaling Technology, catalog number: 3936 )
  16. Goat anti-mouse antibody conjugated to horseradish peroxidase (HRP) (Bio-Rad Laboratories, catalog number: 170-6516 )
  17. Amersham ECL prime Western blotting detection reagent (GE Healthcare, catalog number: RPN2232 )
  18. NaCl
  19. KCl
  20. Na2HPO4
  21. KH2PO4
  22. Tween-20 (Sigma-Aldrich, catalog number: P1379 )
  23. Tris base
  24. Glycine
  25. Methanol
  26. Dialysis buffer (see Recipes)
  27. 1x PBS (see Recipes)
  28. 1x PBST (see Recipes)
  29. Transfer buffer (see Recipes)

Equipment

  1. Incubator (VWR, model number: 1545 ) or water bath
  2. Protein electrophoresis apparatus (Bio-Rad Laboratories, model: Mini PROTEAN® 3 Cell )
  3. Western blotting apparatus (Bio-Rad Laboratories, model: Mini Trans-Blot® Cell )
  4. X-Ray film processor (PROTEC, model: OPTIMAX )

Procedure

  1. Purify 6x His-tagged Arabidopsis E2 UBC22 (His-UBC22) using QIAGEN Ni-NTA Spin Kit and following the manufacturer’s instructions. Purified protein is then dialyzed in a dialysis buffer for overnight.
  2. Prepare the reaction samples in 1.5 ml polypropylene tubes. Different components are added as listed below. To test specific linkage of dimer, wild-type Ub can be substituted by different Ub mutants, each of which has one particular lysine residue mutated (Ub-K11R, Ub-K48R or Ub-K63R).
    Component                  Amount to add or final concentration
    10x buffer                    2.0 μl
    E1                               0.2 μM
    E2                               5.0 μM
    Ub                              62.5 μM
    Mg-ATP solution        1.0 mM
    ddH2O                         make up to 20 μl
  3. Incubate the reaction samples at 30 °C either in a water bath or an incubator for 4 h.
  4. Add 7 μl 4x SDS non-reducing loading buffer and 1 μl ddH2O into each reaction sample.
  5. Treat the samples at 95 °C for 5 min.
  6. Bio-Rad Mini PROTEAN® 3 Cell gel apparatus is used for protein electrophoresis. Load half amount of samples on a 15% SDS-PAGE resolving gel with 4% stacking gel and run the gel at 120 V for 130 min to separate the proteins.
  7. Transfer the proteins to a piece of PVDF membrane at 120 V for 120 min using Bio-Rad Mini Trans-Blot® Cell. The transfer apparatus is placed into a bucket with ice to reduce heat production during the transfer.
  8. Incubate the membrane in 10 ml of 1x PBS containing 5% skimmed milk powder for 0.5 h at room temperature for the blocking treatment.
  9. Incubate the membrane with primary antibody (anti-ubiquitin antibody, antibody dilution ratio: 1:40,000) in 10 ml 1x PBST containing 3% skimmed milk powder overnight at 4 °C.
  10. Wash the membrane with 1x PBST for three times at room temperature (20 ml, 10 min each).
  11. Incubate the membrane with secondary antibody (goat anti-mouse antibody conjugated to HRP, antibody dilution ratio: 1:7,000) in 1x PBST containing 3% skimmed milk powder for 1 h at room temperature.
  12. Wash the membrane with 1x PBST for three times at room temperature (20 ml, 10 min each).
  13. Detect signal with the ECL prime Western blotting detection reagent according to the manufacturer’s instructions (Figure 1). Use 1-10 min as the initial range of film exposure time, depending on signal strength.


    Figure 1. Ub dimer formation assay by His-UBC22. To determine the activity of Arabidopsis UBC22, various components, as indicated on the top of the figure, were added to the reaction tubes with (lanes 2, 4, 6 and 8) or without His-UBC22 (lanes 1, 3, 5 and 7). The reaction tubes were incubated at 30 °C for 4 h. The samples were then subjected to SDS-PAGE (with 4% stacking and 15% resolving gel).Free Ub and Ub dimers were detected by Western blotting using an anti-Ub antibody. Free Ub (Ub) and Ub dimers (Ub2) are indicated on the right side of the figure. For the K11R mutant, no new Ub dimer formation was observed when His-UBC22 protein was added. Due to slight impurity, there was one weak band in the recombinant K11R protein which is slightly higher than the Ub dimer synthesized from the wild-type Ub and other Ub mutants. Three independent experiments were performed and had similar results (Figure from Wang et al., 2016; Figure 6 in the manuscript: http://jxb.oxfordjournals.org/content/early/2016/04/10/jxb.erw142.full).

Data analysis

Purified Arabidopsis recombinant E2 UBC22 fused with 6x His tag (His-UBC22) was tested in the in vitro Ub dimer formation assay. As shown in Figure 1, Ub dimers could be detected when His-UBC22 was added into the reaction (lane 2 compared to the lane 1), indicating the biochemical activity of UBC22 catalyzing free dimer formation. In addition, similar dimer formation was observed when the Ub-K48R mutant or Ub-K63R mutant was used, which lacks K48 residue or K63 residue (lane 6 compared to lane 5, or lane 8 compared to lane 7). Interestingly, when the Ub-K11R which lacks K11 residue was used, little dimer was produced (lane 4 compared to lane 3). These results indicate that UBC22 could catalyze Ub dimer formation in vitro specifically through K11 residue of Ub. Three independent experiments were performed and produced similar results.

Notes

  1. This assay aims to investigate a specific activity of the E2 enzyme. The purity of the recombinant protein or any conditions affecting a protein’s activity could affect the result. A commercial E2 protein, such as Human E2 Ube2S, may be good for a positive control.
  2. Poly-Ub chains might be formed and detected in the assay depending on the activity of an E2 tested.

Recipes

  1. Dialysis buffer
    10 mM Tris-HCl (pH 7.5)
    50 mM NaCl
  2. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4
    Adjust to pH 7.4
  3. 1x PBST
    0.1% Tween-20 in 1x PBS
  4. Transfer buffer
    25 mM Tris base
    192 mM glycine
    20% (v/v) methanol

Acknowledgments

We gratefully acknowledge the financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC) (Discovery grant) to HW.

References

  1. Wang, S., Cao, L. and Wang, H. (2016). Arabidopsis ubiquitin-conjugating enzyme UBC22 is required for female gametophyte development and likely involved in Lys11-linked ubiquitination. J Exp Bot 67(11): 3277-3288.

简介

蛋白质泛素化的过程通常包括三个顺序步骤:向底物蛋白添加泛素(Ub)或Ub链,需要三种不同的酶,泛素活化酶(E1),泛素缀合酶(E2)和泛素蛋白连接酶E3)。大多数E2在E1和Ub的存在下具有通过硫酯键形成E2-Ub复合物的经典E2活性。另外,一些E2具有催化游离Ub二聚体形成的能力。这种活性表明这些E2在泛素化途径中的重要作用。因此,我们开发了体外的Ub二聚体形成测定来确定某些E2的活性。此外,通过使用不同赖氨酸残基突变的Ub突变体,也可以确定二聚体的特异性连接。

背景 E2共轭启动试验(不添加E3和底物)的现有方案旨在检测硫酯键(E2-S-Ub)。我们的方法着重于催化游离Ub二聚体形成(Ub-Ub)的E2活性。提供了一种检测不同物种E2重要生化特征的便捷方式。此外,二聚体的特异性连接可以通过使用不同的Ub突变体来确定。

关键字:泛素化, 泛素二聚体形成, E2, 拟南芥UBC22, K11连接

材料和试剂

  1. 1.5ml聚丙烯管
  2. 聚偏二氟乙烯(PVDF)膜(Bio-Rad Laboratories,目录号:162-0177)
  3. AmershamHyperfilm TM ECL(GE Healthcare,目录号:28906836)
  4. 纯化人重组E1(BOSTONBIOCHEM,目录号:K-995)
  5. Qiagen Ni-NTA Spin Kit(QIAGEN,目录号:31314)
  6. 纯化人重组Ub(BOSTONBIOCHEM,目录号:K-995)
  7. 纯化的人重组Ub与突变的赖氨酸11(K11)残基(Ub-K11R)(BOSTONBIOCHEM,目录号:UM-K11R)
  8. 纯化的人重组Ub与突变的赖氨酸48(K48)残基(Ub-K48R)(BOSTONBIOCHEM,目录号:UM-K48R)
  9. 用Ub(K63)残基突变(Ub-K63R)(BOSTONBIOCHEM,目录号:UM-K63R)的纯化人重组Ub
  10. 10倍反应缓冲液(BOSTONBIOCHEM,目录号:K-995)
  11. Mg-ATP溶液(BOSTONBIOCHEM,目录号:K-995)
  12. 4x非还原加载缓冲区(BOSTONBIOCHEM,目录号:K-995)
  13. SDS-PAGE凝胶
  14. 脱脂奶粉
  15. 抗泛素抗体(Cell Signaling Technology,目录号:3936)
  16. 与辣根过氧化物酶(HRP)缀合的山羊抗小鼠抗体(Bio-Rad Laboratories,目录号:170-6516)
  17. Amersham ECL主要蛋白质印迹检测试剂(GE Healthcare,目录号:RPN2232)
  18. NaCl
  19. KCl
  20. Na 2 HPO 4
  21. KH 2 PO 4
  22. 吐温-20(Sigma-Aldrich,目录号:P1379)
  23. 三碱基
  24. 大豆
  25. 甲醇
  26. 透析缓冲液(见配方)
  27. 1x PBS(见食谱)
  28. 1x PBST(见配方)
  29. 转移缓冲区(见配方)

设备

  1. 孵化器(VWR,型号:1545)或水浴
  2. 蛋白质电泳装置(Bio-Rad Laboratories,型号:Mini PROTEAN 3细胞)
  3. Western印迹装置(Bio-Rad Laboratories,型号:Mini Trans-Blot Cell)
  4. X光片处理器(PROTEC,型号:OPTIMAX)

程序

  1. 使用QIAGEN Ni-NTA Spin Kit纯化6x His标签的拟南芥E2 UBC22(His-UBC22),并按照制造商的说明进行。然后将纯化的蛋白质在透析缓冲液中透析过夜。
  2. 在1.5ml聚丙烯管中制备反应样品。添加不同的组件,如下所示。为了测试二聚体的特异性连接,野生型Ub可以被不同的Ub突变体取代,每个Ub突变体都具有突变的一个特定赖氨酸残基(Ub-K11R,Ub-K48R或Ub-K63R)。
    组件                 添加量或最终浓度
    10倍缓冲区                     2.0μl
    E1                          ;      0.2μM
    E2                          ;     5.0μM
    Ub                          ;     62.5μM
    Mg-ATP溶液        1.0 mM
    ddH 2 O                         补充20μl
  3. 将反应样品在30℃下在水浴或培养箱中孵育4小时。
  4. 在每个反应样品中加入7μl4x SDS非还原加载缓冲液和1μlddH 2 O。
  5. 在95℃下处理样品5分钟。
  6. Bio-Rad Mini PROTEAN ® 3细胞凝胶仪用于蛋白质电泳。在15%SDS-PAGE分解凝胶上加载半量样品,并用4%堆叠凝胶,并在120V下运行凝胶130分钟,以分离蛋白质。
  7. 使用Bio-Rad Mini Trans-Blot Cell将蛋白质在120V下转移到一片PVDF膜上120分钟。转移装置放入带有冰的桶中,以减少转移过程中的热量产生
  8. 在10 ml含有5%脱脂奶粉的1x PBS中孵育0.5小时,以进行封闭治疗。
  9. 在含有3%脱脂奶粉的10ml 1x PBST中,在4℃下将膜与一抗(抗泛素抗体,抗体稀释比为1:40,000)孵育过夜。
  10. 在室温下,用1×PBST洗涤膜3次(20ml,每次10分钟)
  11. 在含有3%脱脂奶粉的1×PBST中室温孵育1小时的二抗(羊抗鼠抗体缀合抗体,抗体稀释比为1:7,000)的膜。
  12. 在室温下,用1×PBST洗涤膜3次(20ml,每次10分钟)
  13. 根据制造商的说明书(图1),用ECL主要蛋白质印迹检测试剂检测信号。使用1-10分钟作为胶片曝光时间的初始范围,具体取决于信号强度。


    图1.通过His-UBC22进行的Ub二聚体形成测定为了确定拟南芥UBC22的活性,将如图顶部所示的各种成分加入到具有(泳道2,4,6和8)或不含His-UBC22(泳道1,3,5和7)的反应管。将反应管在30℃下孵育4小时。然后将样品进行SDS-PAGE(具有4%堆积和15%分辨凝胶)。通过使用抗Ub抗体的Western印迹检测到Ub和Ub二聚体。在图的右侧表示免费的Ub(Ub)和Ub二聚体(Ub 2+)。对于K11R突变体,当添加His-UBC22蛋白时,没有观察到新的Ub二聚体形成。由于轻微的杂质,重组K11R蛋白中有一个弱带,略高于从野生型Ub和其他Ub突变体合成的Ub二聚体。执行了三个独立的实验,具有相似的结果(Wang等人,2016年的图;图6在手稿中: http://jxb.oxfordjournals.org/content/early/2016/04/10/jxb.erw142。完整)。

数据分析

纯化的拟南芥在体外Ub二聚体形成测定中测试与6x His标签(His-UBC22)融合的重组E2 UBC22。如图1所示,当将His-UBC22加入反应(泳道2与泳道1相比)时,可以检测到Ub二聚体,表明UBC22催化游离二聚体形成的生化活性。此外,当使用Ub-K48R突变体或Ub-K63R突变体时,观察到类似的二聚体形成,其缺少K48残基或K63残基(泳道5与泳道5相比,或泳道8与泳道7相比)。有趣的是,当使用缺乏K11残基的Ub-K11R时,产生很少的二聚体(与泳道3相比,泳道4)。这些结果表明,UBC22可以通过Ub的K11残基特异性地在体外催化Ub二聚体形成。进行了三次独立实验,得到了类似的结果。

笔记

  1. 该测定旨在研究E2酶的比活性。重组蛋白的纯度或影响蛋白质活性的任何条件都可能影响结果。商业E2蛋白,如人E2 Ube2S,可能对阳性对照有好处。
  2. 根据所测试的E2的活性,可以在测定中形成和检测聚-UP链。

食谱

  1. 透析缓冲液
    10mM Tris-HCl(pH7.5)
    50 mM NaCl
  2. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4
    1.8mM KH PO 4
    调整至pH 7.4
  3. 1x PBST
    0.1%Tween-20在1x PBS中
  4. 转移缓冲区
    25 mM Tris碱基
    192mM甘氨酸
    20%(v/v)甲醇

致谢

我们非常感谢加拿大自然科学和工程研究理事会(NSERC)(发现资助)向HW提供财务支持。

参考

  1. Wang,S.,Cao,L.and Wang,H。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27069118"目标="_ blank">拟南芥泛素结合酶UBC22是雌配子体发育所必需的,可能涉及Lys11连锁的泛素化。 J Exp Bot 67(11):3277-3288。
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引用:Wang, S., Cao, L. and Wang, H. (2017). In vitro Ubiquitin Dimer Formation Assay. Bio-protocol 7(1): e2082. DOI: 10.21769/BioProtoc.2082.
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