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Acid Extraction of Total Histone from Colon Cancer HCT116 Cells
酸性法提取结肠癌细胞HCT116的组蛋白   

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Abstract

Histone acid extraction assay is a popular method to determine histone modification levels in mammalian cells. It includes three steps: first, histones are released from chromatin by sulfuric acid; trichloroacetate (TCA) is then added to precipitate histones; and finally, histones are dissolved in double-distilled H2O (ddH2O). Here we present a detailed histone acid extraction assay in our laboratory using a colon cancer cell line, HCT116, as a model.

Background

The nucleosome is the fundamental unit of eukaryotic chromatin, which is composed of a histone octamer (2 copies of H3, H4, H2A, H2B, respectively) wrapping by DNA (Strahl and Allis, 2000). The amino terminal of histone is subjected to a variety of post-translational modifications, such as methylation, acetylation, phosphorylation, ubiquitylation and sumoylation (Kouzarides, 2007). Although the function of these modifications has remained elusive, there is ever-growing studies suggest that histone modifications play vital roles in intracellular processes (Bannister and Kouzarides, 2011). Therefore, it is important to extract histones efficiently to detect histone modifications.

Histones can be extracted via different methods, in which histone acid extraction assay is one of the most popular procedures. It does not interrupt post-translational modifications of histones, and so it is very good for histone modification analysis. It has been tested that the extracted histones can be used in Western blot, and maybe other assays (not fully tested). However, immunoprecipitation is not recommended. In this protocol, we will present a detailed histone acid extraction assay, and describe how to release histones from chromatin, how to precipitate histones, and how to wash and dissolve histones in ddH2O.

Materials and Reagents

  1. 6 cm plate
  2. 1.5 ml tubes (Corning, Axygen®, catalog number: MCT-150-C )
  3. Human colon cancer cell line HCT116 (ATCC)
  4. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM)
  5. Acetone
  6. Double-distilled H2O (ddH2O)
  7. 2x SDS loading buffer (containing 200 mmol/L DTT)
  8. Tris-HCl (pH = 8.0)
  9. Potassium chloride (KCl)
  10. Magnesium chloride (MgCl2)
  11. Dithiothreitol (DTT)
  12. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
  13. Sulfuric acid
  14. Trichloroacetate (TCA) (Sigma-Aldrich, catalog number: T6399 )
  15. Trichloroacetic acid (TCA) solution (see Recipes)
  16. Lysis buffer(see Recipes)

Equipment

  1. Pipettor (Eppendorf)
  2. Centrifuge (cooled and room-temperature)(Eppendorf)
  3. Rotator
  4. Spectrophotometer (Bio-Rad Laboratories)
  5. Metal bath

Procedure

An overview of the whole procedure is schematized in Figure 1.


Figure 1. Scheme of the complete procedure described in the protocol

  1. Collect HCT116 cells (6 cm plate, 70-90% confluent) using any commonly used method in 1 ml ice-cold PBS and pellet (1,000 x g, 5 min, 4 °C).
  2. Discard the supernatant, add 1 ml ice-cold PBS and pipet gently to resuspend the cells, then pellet it again (1,000 x g, 5 min, 4 °C).
  3. Discard the supernatant, resuspend cell pellet in 400 μl lysis buffer, and incubate for 30 min with rotating at 4 °C.
  4. Pellet the intact nuclei by spinning in cooled centrifuge (12,000 x g, 10 min, 4 °C).
  5. Transfer the supernatant containing histones into a fresh 1.5 ml tube.
  6. Add 133 μl TCA drop by drop to histone solution and invert the tube several times to mix the solutions (final concentration of TCA is 25%). (It is better to add one drop and invert the tube at once to mix the solutions, and then add another drop.) The solution will appear milky over time.
  7. Incubate on ice for 30 min or overnight.
  8. Pellet histones by spinning in cooled centrifuge (12,000 x g, 10min, 4 °C).
  9. Carefully remove supernatant and wash histone pellet with 1 ml ice cold acetone without disturbing it. Acetone is used to remove acid from the solution without dissolving the histone pellet.
  10. Spin down in centrifuge at 12,000 x g, 10 min at 4 °C.
  11. Repeat steps 9 and 10 to wash histone pellet again.
  12. Carefully remove all of the supernatant and air-dry histone pellet for 30 min at room temperature.
  13. Dissolve histone pellet in appropriate volume of ddH2O (for example, 100 μl is appropriate for a 6 cm plate), and transfer into fresh 1.5 ml tube.
  14. Detect the concentration of histone solution with the spectrophotometer (using OD 280), and we may acquire 1-2 µg histone protein from a 70-90% confluent well on a 6 cm plate.
  15. Add the same volume of 2x SDS loading buffer into histone solution, and boil it on the metal bath (100 °C, 5 min), then subject to Western blot.

Representative data


Figure 2. HCT116 cells were treated with etoposide and subjected to histone acid extraction. Immunoblotting was performed with the indicated antibodies.

Note: Relative Western blots have also been published in our paper Oncogene (Cao et al., 2016). For details, please refer to Figure 4 in Cao et al., 2016 (https://www.researchgate.net/publication /274316705_ATM-mediated_KDM2A_phosphorylation_is_required_for_the_DNA_damage_repair).

Recipes

  1. Trichloroacetic acid (TCA) solution
    500 g TCA in 227 ml ddH2O
    Store at room temperature, and protect from light
  2. Lysis buffer
    10 mM Tris-HCl, pH 8.0
    1 mM KCl
    1.5 mM MgCl2
    10 mM DTT (added immediately before use)
    1x protease inhibitor cocktail (added immediately before use)
    0.4 M sulfuric acid (added immediately before use)

Acknowledgments

This work was supported by Beijing Natural Science Foundation grant 7164305, the ‘973 Projects’ (2011CB910100, 2011CB504200 and 2013CB911000), National Natural Science Foundation of China (81222028, 81321003, 81472581, 81530074, 31570812 and 91319302), and grants (B70001) from the Ministry of Science and Technology of China. This protocol was adapted from previous work published in Cancer Research (Zhu et al., 2001), Oncogene (Cao et al., 2016) and Oncotarget (Lu et al., 2015).

References

  1. Bannister, A. J. and Kouzarides, T. (2011). Regulation of chromatin by histone modifications. Cell Res 21(3): 381-395.
  2. Cao, L. L., Wei, F., Du, Y., Song, B., Wang, D., Shen, C., Lu, X., Cao, Z., Yang, Q., Gao, Y., Wang, L., Zhao, Y., Wang, H., Yang, Y. and Zhu, W. G. (2016). ATM-mediated KDM2A phosphorylation is required for the DNA damage repair. Oncogene 35: 301-313.
  3. Kouzarides T. (2007). Chromatin modifications and their function. Cell 128: 693-705.
  4. Lu, S., Yang, Y., Du, Y., Cao, L. L., Li, M., Shen, C., Hou, T., Zhao, Y., Wang, H., Deng, D., Wang, L., He, Q. and Zhu, W. G. (2015). The transcription factor c-Fos coordinates with histone lysine-specific demethylase 2A to activate the expression of cyclooxygenase-2. Oncotarget 6(33): 34704-34717.
  5. Strahl, B. D. and Allis, C. D. (2000). The language of covalent histone modifications. Nature 403(6765): 41-45.
  6. Zhu, W. G., Lakshmanan, R. R., Beal, M. D. and Otterson, G. A. (2001). DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors. Cancer Res 61(4): 1327-1333.

简介

组蛋白酸提取测定法是确定哺乳动物细胞中组蛋白修饰水平的常用方法。它包括三个步骤:第一,组蛋白通过硫酸从染色质释放;然后加入三氯乙酸(TCA)以沉淀组蛋白;最后,将组蛋白溶解于双蒸H 2 O(ddH 2 O)。在这里我们提出一个详细的组蛋白酸提取测定法在我们的实验室使用结肠癌细胞系,HCT116,作为一个模型。

[背景] 核小体是真核染色质的基本单元,其由组蛋白八聚体(分别为H3,H4,H2A,H2B的2个拷贝)组成通过DNA(Strahl和Allis,2000)。组蛋白的氨基末端经受各种翻译后修饰,例如甲基化,乙酰化,磷酸化,泛素化和sumoylation(Kouzarides,2007)。虽然这些修改的功能仍然难以捉摸,但越来越多的研究表明组蛋白修饰在细胞内过程中起着至关重要的作用(Bannister和Kouzarides,2011)。因此,重要的是有效地提取组蛋白以检测组蛋白修饰。  组蛋白可以通过不同的方法提取,其中组蛋白酸提取测定是最流行的程序之一。它不会中断组蛋白的翻译后修饰,因此它对组蛋白修饰分析非常有用。已经测试提取的组蛋白可以用于Western印迹,以及可能的其他测定(未完全测试)。然而,不建议免疫沉淀。在该协议中,我们将提出详细的组蛋白酸提取测定,并描述如何从染色质释放组蛋白,如何沉淀组蛋白,以及如何清洗和溶解组蛋白在ddH 2 O中。

材料和试剂

  1. 6厘米板
  2. 1.5ml管(Corning,Axygen ,目录号:MCT-150-C)
  3. 人结肠癌细胞系HCT116(ATCC)
  4. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco< sup> TM)
  5. 丙酮
  6. 双蒸H 2 O(ddH 2 O)
  7. 2x SDS上样缓冲液(含有200mmol/L DTT)
  8. Tris-HCl(pH = 8.0)
  9. 氯化钾(KCl)
  10. 氯化镁(MgCl 2)
  11. 二硫苏糖醇(DTT)
  12. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)
  13. 硫酸
  14. 三氯乙酸酯(TCA)(Sigma-Aldrich,目录号:T6399)
  15. 三氯乙酸(TCA)溶液(参见配方)
  16. 裂解缓冲液(见配方)

设备

  1. Pipettor(Eppendorf)
  2. 离心(冷却和室温)(Eppendorf)
  3. 旋转器
  4. 分光光度计(Bio-Rad Laboratories)
  5. 金属浴

程序

整个过程的概述如图1所示。

图1.在协议
中描述的完整过程的方案

  1. 使用任何常用的方法在1ml冰冷的PBS和沉淀(1000×g,5分钟,4℃)中收集HCT116细胞(6cm平板,70-90%汇合)。< br/>
  2. 弃去上清液,加入1ml冰冷的PBS,轻轻吸取以重悬细胞,然后再次沉淀(1,000×g,5分钟,4℃)。
  3. 弃去上清液,将细胞沉淀重悬在400μl裂解缓冲液中,并在4℃下旋转孵育30分钟。
  4. 通过在冷却的离心机(12,000×g,10分钟,4℃)中旋转来制粒完整的核。
  5. 将含有组蛋白的上清液转移到新鲜的1.5ml管中
  6. 逐滴加入133微升TCA到组蛋白溶液中,倒置管数次以混合溶液(TCA的最终浓度为25%)。 (最好添加一滴并立即倒转管以混合溶液,然后再添加一滴)。溶液随时间出现乳白色。
  7. 在冰上孵育30分钟或过夜。
  8. 通过在冷却的离心机(12,000×g,10分钟,4℃)中旋转来沉淀组织蛋白。
  9. 小心除去上清液和用1ml冰冷的丙酮洗涤组蛋白沉淀,而不打扰它。丙酮用于从溶液中除去酸而不溶解组蛋白沉淀
  10. 在离心机中以12,000×g离心,在4℃下离心10分钟
  11. 重复步骤9和10,再次洗涤组蛋白沉淀
  12. 在室温下小心地除去所有的上清液和空气干燥的组蛋白沉淀30分钟
  13. 将组蛋白沉淀溶解在适当体积的ddH 2 O(例如,100μl适合于6cm平板)中,并转移到新鲜的1.5ml管中。
  14. 用分光光度计(使用OD 280)检测组蛋白溶液的浓度,我们可以从6cm平板上的70-90%汇合孔获得1-2μg组蛋白。
  15. 加入相同体积的2x SDS加载缓冲液到组蛋白溶液中,并在金属浴(100℃,5分钟)煮沸,然后进行蛋白质印迹。

代表数据



图2.用依托泊苷处理HCT116细胞并进行组蛋白酸提取。用指定的抗体进行免疫印迹。

注意:相对的Western印迹也已经在我们的论文Oncogene(Cao等人,2016)中发表。有关详细信息,请参阅Cao等人,2016年的图4( https://www.researchgate.net/publication/274316705_ATM-mediated_KDM2A_phosphorylation_is_required_for_the_DNA_damage_repair )。

食谱

  1. 三氯乙酸(TCA)溶液
    在227ml ddH 2 O中的500g TCA 储存在室温下,避光
  2. 裂解缓冲液
    10mM Tris-HCl,pH8.0 1 mM KCl
    1.5mM MgCl 2·h/v 10mM DTT(使用前立即加入)
    1x蛋白酶抑制剂混合物(在使用前立即加入)
    0.4 M硫酸(使用前立即加入)

致谢

这项工作得到了北京市自然科学基金资助项目7164305,国家自然科学基金(81222028,81321003,81472581,81530074,31570812和91319302)的'973项目'(2011CB910100,2011CB504200和2013CB911000)和资助项目(B70001)来自中国科学技术部。该方案改编自以前在Cancer Research(Zhu等人,2001),Oncogene(Cao等人,2016)和Oncotarget(Lu等人)中公开的工作, et al。, 2015)。

参考文献

  1. Bannister,AJ和Kouzarides,T。(2011)。  通过组蛋白修饰调节染色质。 Cell Res 21(3):381-395。
  2. Cao,LL,Wei,F.,Du,Y.,Song,B.,Wang,D.,Shen,C.,Lu,X.,Cao,Z.,Yang,Q.,Gao, ,L.,Zhao,Y.,Wang,H.,Yang,Y. and Zhu,WG(2016)。  ATM介导的KDM2A磷酸化是DNA损伤修复所必需的。癌基因 35:301-313。
  3. Kouzarides T.(2007)。  染色质修饰及其功能。 128:693-705。
  4. Lu,S.,Yang,Y.,Du,Y.,Cao,LL,Li,M.,Shen,C.,Hou,T.,Zhao,Y.,Wang,H.,Deng,D.,Wang ,L.,He,Q.and Zhu,WG(2015)。  转录因子c-Fos与组蛋白赖氨酸特异性脱甲基酶2A配位以激活环氧合酶-2的表达。 Oncotarget 6(33) :34704-34717。
  5. Strahl,BD和Allis,CD(2000)。  403(6765):41-45。
  6. Zhu,W.G.,Lakshmanan,R.R.,Beal,M.D.and Otterson,G.A。(2001)。 DNA甲基转移酶抑制增强由组蛋白脱乙酰酶抑制剂诱导的凋亡。  Cancer Res 61(4):1327-1333
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cao, L. and Zhu, W. (2016). Acid Extraction of Total Histone from Colon Cancer HCT116 Cells. Bio-protocol 6(22): e2023. DOI: 10.21769/BioProtoc.2023.
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