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Ubiquitination Assay for Mammalian Cells
哺乳动物细胞的泛素化试验   

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Abstract

Ubiquitin is an 8.5 kDa protein that can be activated and conjugated by ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, respectively. Ubiquitin E3 ligases then recognize protein substrates, and then transfer the ubiquitin from E2 to the targeted protein. This biological process is called ubiquitination, and it is an important biological process which can signal protein degradation via the proteasome. The aim of this protocol is to describe a procedure that determines the level of cellular ubiquitination in a protein-of-interest relative to control cells.

Keywords: Ubiquitination(泛素化), Mammalian Cells(哺乳动物细胞), in vivo(在体内)

Materials and Reagents

  1. 10 cm dish
  2. Microcentrifuge tubes
  3. Eppendorf tubes
  4. Agarose beads
  5. U2OS cells
  6. HA-Ubiquitin plasmid
  7. FuGene 6 transfection reagent (Promega Corporation, FuGENE®, catalog number: E2691 )
  8. Reduced-serum medium (Thermo Fisher Scientific, Gibco®, catalog number: 31985-070 )
  9. MG132
  10. 1x PBS
  11. Trypsin (Thermo Fisher Scientific, Gibco®, catalog number: 25300 )
  12. IgG control antibody according to the targeted antibody species [e.g., Rabbit IgG (Santa Cruz Biotechnology, catalog number: sc-2027 )]
  13. Agarose conjugate [e.g., Protein A/G agarose (Santa Cruz Biotechnology, catalog number: sc-2003 )]
  14. BRCA1 antibody
  15. RIPA buffer (e.g., Santa Cruz Biotechnology, catalog number: sc-24948 )
  16. Anti-ubiquitin or anti-HA antibody
  17. Tris-HCl
  18. SDS
  19. Bromophenol blue
  20. β-mercaptoethanol
  21. Glycerol
  22. Loading buffer (see Recipes)

Equipment

  1. Tube rotator located in cold room 4 °C
  2. Centrifuges (Eppendorf, model: 5424/5424 R )

Procedure

  1. Co-transfect U2OS cells with HA-Ubiquitin plasmid with other plasmids of interest using FuGene 6. For a 10 cm dish, 3 μg of plasmid is mixed with 18 μl of Fugene 6 in 500 μl Reduced-serum medium, following the manufacturer's recommended procedure for use of Fugene 6. In the representative image, TUSC4 knockdown U2OS cells were used.
  2. After 15-30 min of incubation of the mixture at room temperature, add the mixture drop-wise onto the cell culture media of the 10 cm dish. Gently move the dish around to spread the mixture evenly throughout the dish.
  3. Harvest the cells within 48-72 h (10 µM MG132 incubation for 4-6 h prior to harvest is an optional step that enriches the ubiquitination signal).
  4. Prepare total cell lysate as described under a standard Western blot procedure. In short, wash the cells with 1x PBS and incubate cells with a volume of trypsin that is sufficient to detach the cells from the dish (e.g., 200 µl). Lyse the cells directly on the dish using RIPA buffer (e.g., 500 µl), then transfer the cells to a microcentrifuge tube.
  5. Disrupt the cells with gentle sonication (e.g., 3-sec pulse for 5 times).
  6. Centrifuge the sample at 10,000 x g for 10 min at 4 °C, then transfer the supernatant to a clean microcentrifuge tube on ice, without transferring the cellular debris at the bottom.
  7. (Optional step to reduce non-specific binding) Preclear cell lysate with appropriate IgG mouse control antibody (e.g., 0.25 µg antibody per 1 ml cell lysate), and 20 μl suspended agarose conjugate (e.g., Protein A/G agarose) and rotate mixture at 4 °C for 30 min to 1 h.
  8. Spin down the mixture at 1,000 x g for 1 min at 4 °C and transfer the supernantant containing the targeted protein into a clean microcentrifuge tube (a typical amount of protein is 2-5 mg). Incubate samples with an amount of primary antibody that is suggested by the manufacture at 4 °C with rotation for 2 h to overnight. In the representative image, BRCA1 antibody was used (Lu et al., 2012).
  9. Add 20 µl of appropriate agarose beads and incubate the samples at 4 °C with rotation for 1-2 h.
  10. Spin down the agarose beads at 1,000 x g for 1 min at 4 °C, discard the supernatant with care to not disturb the beads.
  11. Wash the beads with RIPA buffer or PBS 3 times with constant rotation at 4 °C, 15 min each time. RIPA buffer is a relatively strong detergent which can disrupt protein-protein interactions; alternate detergents may be considered for weaker protein-protein interactions. After the last wash, remove as much buffer as possible with minimal disruption of the beads.
    Note: In some case, RIPA would be too harsh, so this needs to be adjusted case by case.
  12. Add 20-50 µl of loading buffer and mix to re-suspend the beads. Incubate the samples for 5 min at 95 °C, and then subject to SDS-PAGE analysis or store the samples in -20 °C for future use. In the representative image, a 10% gel was used. Regular western blot condition is sufficient; some blots may require extended wash due to high background that can be caused by non-specific binding during the immunoprecipitation step.
  13. Blots can be probed using anti-ubiquitin or anti-HA antibody to determine the primary proteins’ ubiquitination levels.

Representative data


Figure 1. Knockdown of TUSC4 expression enhanced BRCA1 polyubiquitination, leading to BRCA1 protein degradation

NPRL2/TUSC4 knockdown increased ubiquitination level of BRCA1 compared with control cells (after MG132 enrichment for ubiquitination) (Peng et al., 2015).

Notes

  1. Preclear cell lysate with appropriate IgG control antibody is a necessary step to reduce background signals.

Recipes

  1. Loading buffer
    62.5 mM Tris-HCl (pH 6.8)
    2.5% SDS
    0.002% bromophenol blue
    0.7135 M (5%) β-mercaptoethanol
    10% glycerol

Acknowledgments

This work was supported by The Department of Defense (DOD) Breast Cancer Research Program (BCRP) Era of Hope Scholar Award (W81XWH-10-1-0558; S.-Y. Lin).

References

  1. Lu, Y., Li, J., Cheng, D., Parameswaren, B., Zhang, S., Jiang, Z.,Yew, P. R., Peng, J., Ye, Q., Hu, Y. (2012). The F-box protein FBXO44 mediates BRCA1 ubiquitination and degradation. J Biol Chem 287:41014-22.
  2. Peng, Y., Dai, H., Wang, E., Lin, C.C., Mo, W., Peng, G., Lin, S.Y. (2015). TUSC4 Functions as a Tumor Suppressor by Regulating BRCA1 Stability. Cancer Res 75(2):378-86.

简介

泛素是8.5kDa的蛋白质,其可以分别由泛素激活酶E1和泛素缀合酶E2激活和缀合。 然后泛素E3连接酶识别蛋白质底物,然后将泛素从E2转移到目标蛋白质。 这种生物过程称为泛素化,它是一个重要的生物过程,可以通过蛋白酶体信号蛋白降解。 该方案的目的是描述确定相对于对照细胞的目的蛋白质中的细胞泛素化水平的程序。

关键字:泛素化, 哺乳动物细胞, 在体内

材料和试剂

  1. 10厘米培养皿
  2. 微量离心管
  3. Eppendorf管
  4. 琼脂糖珠
  5. U2OS细胞
  6. HA-遍在蛋白质粒
  7. FuGene 6转染试剂(Promega Corporation,FuGENE ,目录号:E2691)
  8. 还原血清培养基(Thermo Fisher Scientific,Gibco ,目录号:31985-070)
  9. MG132
  10. 1x PBS
  11. 胰蛋白酶(Thermo Fisher Scientific,Gibco ,目录号:25300)
  12. IgG对照抗体根据靶向抗体种类[例如,兔IgG(Santa Cruz Biotechnology,目录号:sc-2027)]/
  13. 琼脂糖缀合物[例如,Protein A/G琼脂糖(Santa Cruz Biotechnology,目录号:sc-2003)]
  14. BRCA1抗体
  15. RIPA缓冲液(例如,Santa Cruz Biotechnology,目录号:sc-24948)
  16. 抗遍在蛋白或抗HA抗体
  17. Tris-HCl
  18. SDS
  19. 溴酚蓝
  20. β-巯基乙醇
  21. 甘油
  22. 加载缓冲区(参见配方)

设备

  1. 管式旋转器位于4℃的冷室
  2. 离心机(Eppendorf,型号:5424/5424R)

程序

  1. 使用FuGene 6用HA-泛素质粒与其它感兴趣的质粒共转染U2OS细胞。对于10cm培养皿,将3μg质粒与18μlFugene 6在500μl还原型血清培养基中按照制造推荐程序混合用于Fugene 6.在代表性图像中,使用TUSC4敲低U2OS细胞。
  2. 在室温下温育混合物15-30分钟后,将混合物逐滴加入10cm培养皿的细胞培养基上。轻轻移动盘子,将混合物均匀地撒在盘子上
  3. 在48-72小时内收获细胞(10μMMG132孵育4-6小时,收获前是富集泛素化信号的任选步骤)。
  4. 按照标准Western印迹程序所述制备总细胞裂解物。简而言之,用1x PBS洗涤细胞,并用足以从培养皿分离细胞的体积的胰蛋白酶孵育细胞(例如,200μl)。使用RIPA缓冲液(例如,500μl)直接在培养皿上裂解细胞,然后将细胞转移到微量离心管中。
  5. 用温和超声处理(例如,3秒脉冲5次)破坏细胞。
  6. 在4℃下以10,000×g离心样品10分钟,然后将上清液转移到冰上的干??净微量离心管中,而不在底部转移细胞碎片。
  7. (任选的减少非特异性结合的步骤)用合适的IgG小鼠对照抗体(例如,每1ml细胞裂解物中0.25μg抗体)预清洗细胞裂解物和20μl悬浮的琼脂糖缀合物例如Protein A/G琼脂糖),并将混合物在4℃下旋转30分钟至1小时。
  8. 在4℃下以1,000×g离心混合物1分钟,并将含有靶蛋白的上清液转移到干净的微量离心管中(典型量的蛋白质为2-5mg)。孵育样品与一级抗体的量,由制造商建议在4°C旋转2小时过夜。在代表性图像中,使用BRCA1抗体(Lu等人,2012)。
  9. 加入20微升适当的琼脂糖珠,孵化样品在4°C旋转1-2小时
  10. 在4℃下将琼脂糖珠子在1,000×g下旋转1分钟,弃去上清液,小心不要打扰珠子。
  11. 用RIPA缓冲液或PBS洗涤珠子3次,在4℃下恒定旋转,每次15分钟。 RIPA缓冲液是相对强的洗涤剂,其可以破坏蛋白质 - 蛋白质相互作用;可以考虑替代的洗涤剂用于较弱的蛋白质 - 蛋白质相互作用。在最后一次洗涤后,尽可能多地除去珠子的最少破碎的缓冲液。
    注意:在某些情况下,RIPA会过于苛刻,因此需要根据情况进行调整。
  12. 加入20-50微升的加载缓冲液,并混合重新悬浮珠。在95°C孵育样品5分钟,然后进行SDS-PAGE分析或存储在-20°C的样品以备将来使用。在代表性图像中,使用10%凝胶。常规的蛋白质印迹条件是足够的;由于在免疫沉淀步骤期间非特异性结合可能引起的高背景,一些印迹可能需要延长洗涤。
  13. 可以使用抗泛素或抗HA抗体探测印迹以确定初级蛋白的泛素化水平。

代表数据



图1. TUSC4表达的敲低增强了BRCA1多聚泛素化,导致BRCA1蛋白质降解

NPRL2/TUSC4敲低增加了BRCA1与对照细胞(在MG132富集用于泛素化后)的泛素化水平(Peng等人,2015)。

笔记

  1. 用适当的IgG对照抗体预清晰的细胞裂解物是减少背景信号的必要步骤。

食谱

  1. 加载缓冲区
    62.5mM Tris-HCl(pH6.8)
    2.5%SDS
    0.002%溴酚蓝
    0.7135M(5%)β-巯基乙醇 10%甘油

致谢

这项工作是由国防部(DOD)乳腺癌研究计划(BCRP)希望学者时代的支持(W81XWH-10-1-0558; S.-Y.林)。

参考文献

  1. Lu,Y.,Li,J.,Cheng,D.,Parameswaren,B.,Zhang,S.,Jiang,Z.,Yew,PR,Peng,J.,Ye,Q.,Hu, )。  F-box蛋白FBXO44介导BRCA1泛素化,降解。 J Biol Chem 287:41014-22。
  2. Peng,Y.,Dai,H.,Wang,E.,Lin,C.C.,Mo,W.,Peng,G.,Lin, (2015)。  TUSC4通过调节作用作为肿瘤抑制剂BRCA1 Stability。 Cancer Res 75(2):378-86
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Peng, Y., Wang, E., Peng, G. and Lin, S. (2016). Ubiquitination Assay for Mammalian Cells . Bio-protocol 6(14): e1880. DOI: 10.21769/BioProtoc.1880.
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