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Aug 2012
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Subcellular Fractionation of Cultured Human Cell Lines
人源细胞系的亚细胞分离   

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Abstract

Subcellular localization is crucial for the proper functioning of a protein. Deregulation of subcellular localization may lead to pathological consequences and result in diseases like cancer. Immuno-fluorescent staining and subcellular fractionation can be used to determine localization of a protein. Here we discuss a protocol to separate the nuclear, cytosolic, and membrane fractions of cultured human cell lines using a centrifuge and ultracentrifuge. The membrane fraction contains plasma membranes and ER-golgi membranes, but no mitochondria or nuclear structures. The fractions can be further analyzed using Western blotting. This protocol is based on that from Dr. Richard Patten at Abcam, and was modified and utilized in a publication by Huang et al. (2012).

Keywords: Nuclear (细胞核), Cytosolic (细胞溶质的), Membrane (膜), Centrifugation (离心), Fractionation (分馏)

Materials and Reagents

  1. Sucrose
  2. HEPES
  3. Potassium chloride (KCl)
  4. Magnesium chloride (MgCl2)
  5. Ethylene diamine tetraacetic acid (EDTA)
  6. Ethylene glycol tetraacetic acid (EGTA)
  7. Dithiothreitol (DTT)
  8. Tris (Affymetrix-USB, catalog number: 75825 )
  9. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 13565 )
  10. Nonidet P40 substitute (NP40)
  11. Sodium deoxycholate
  12. Glycerol
  13. Sodium dodecyl sulfate (SDS)
  14. Protease inhibitor (PI) cocktails (F. Hoffmann-La Roche, catalog number: 11836145001 )
  15. Methanol
  16. Acetic acid
  17. Brilliant Blue R (Affymetrix, catalog number: 32826 )
  18. Phosphate buffered saline (PBS)
  19. Histone H3 antibody (Cell Signaling Technology, catalog number: 9715 )
  20. Alpha-tubulin antibody (GeneTex, catalog number: GTX108784 )
  21. Cell scraper (BD Biosciences, Falcon®, catalog number: 353086 )
  22. Subcellular fractionation buffer (SF buffer) (see Recipes)
  23. Nuclear Lysis buffer (NL buffer) (see Recipes)
  24. Brilliant Blue R staining solution and destaining solution (see Recipes)

Equipment

  1. 4 °C Microcentrifuge (Eppendorf, catalog number: 5415R );
  2. Ultracentrifuge (Beckman Coulter, model number: Optima TLX )
  3. Optional: Sonicator (Sonics, model number: VC505 )
  4. Whatman filter paper
  5. 37 °C incubator
  6. 1.5 ml Eppendorf microtubes
  7. Tube roller (Maplelab-scientific, model number: MTR-1D )

Procedure

Note: Keep the sample at 4°C on ice at ALL times! All buffers must be ice-cold when used. All centrifugations are done in the Eppendorf Microcentrifuge unless stated otherwise.

  1. Culture cells on 100 mm culture plate until 75% confluent in a 37 °C incubator supplied with 5% CO2. For beginners, the well-studied HEK293 and its derivatives are recommended for easy maintenance and ectopic protein expression.
    Note: This is for adherent cell. Suspension cells may need centrifugation before lysis.
  2. Wash twice with ice-cold PBS and immediately add 500 μl per 100 mm plate of SF buffer and put on ice, use cell scraper to collect lysate and transfer to a 1.5 ml Eppendorf tube. If multiple samples are collected, process one specimen at a time.
  3. Agitate the lysates at 4 °C for 30 min at around 30-50 rpm on the tube roller.
  4. Centrifuge at 720 x g at 4 °C for 5 min. Carefully transfer the supernatant to a new 1.5 ml tube for future use. Keep the pellet for next step.
  5. Wash the pellet with 500 μl of SF buffer and disperse the pellet with a pipette.
  6. Centrifuge the pellet at 720 x g at 4 °C for 10 min.
  7. Remove the supernatant and resuspend the pellet in NL buffer. Agitate and incubate at 4 °C for 15 min.
    Optional: Sonicate the pellet on ice (2 x 3 sec sonication, separated by 3 sec resting, under 30% full amplitude power. On ice!). This is the nuclear fraction including nuclear membranes.
  8. Centrifuge the supernatant from step 4 at 10,000 x g at 4 °C for 10 min.
  9. Carefully transfer the supernatant to a new 1.5 ml tube. This is the cytosolic and membrane fraction.
  10. Centrifuge the cytosolic and membrane fraction from step 9 in an ultracentrifuge. Ultracentrifuge at 100,000 x g at 4 °C for 1 h. Carefully transfer the supernatant to a new 1.5 ml tube. This is the cytosolic fraction. 
  11. Wash the pellet with 500 μl of SF buffer and re-suspend by pipetting.
  12. Ultracentrifuge the pellet at 100,000 x g at 4 °C for 1 h.
  13. Remove the supernatant and re-suspend the pellet in NL buffer.
    Optional: Sonicate the pellet on ice (same setting as for nuclear fraction in step 7). This is the membrane fraction.
  14. Internal loading control for Western blotting could be used to make sure each fraction does not cross-contaminate others; but relative amount can also be determined between samples to ensure equal loading. For example, alpha-tubulin is used for the cytosolic fraction; histone H3 is used for the nuclear fraction; Brilliant Blue R is used for staining for the membrane fraction. Use an extra gel for the loading controls. Alpha-tubulin and histone H3 are probed after protein is transferred onto a PVDF membrane. Brilliant Blue R staining can be applied directly to the SDS-PAGE gel (Figure 1).
    Note: Brilliant Blue R is used here for monitoring relative amount of protein loading across different samples, but is unable to show cross contamination. Membrane proteins such as EGF receptor and integrins may be used to confirm cross-contamination between the membrane and other fractions. However the recycling of these membrane proteins can be an issue, which may appear to be false-positive cross-contamination.


    Figure 1. Western blotting of nuclear, cytoplasmic, and membrane fractions with internal controls. Alpha-tubulin and histone H3 are used for the cytoplasmic and nuclear fractions, respectively; Brilliant Blue R staining is applied for the membrane fraction.

Recipes

  1. Subcellular fractionation buffer (SF buffer)

    Stocks                    
    50 ml 1x solution      
    250 mM Sucrose
    -
    4.28 g
    20 mM HEPES (pH 7.4)
    1 M
    1 ml
    10 mM KCl
    -
    0.0373 g
    1.5 mM MgCl2
    1 M
    75 μl
    1 mM EDTA
    0.5 M
    100 μl
    1 mM EGTA
    0.5 M
    100 μl
  2. At time of use, add the following into 10 ml of SF buffer
                                                   
    Stocks
    10 ml 1x solution                 
    1 mM DTT
    1 M
    10 μl
    PI cocktail
    40x (dissolve 1 tablet in 2 ml dd H2O)
    250 μl
  3. Nuclear Lysis buffer (NL buffer)

    Stocks                        
    50 ml 1x solution          
    50 mM Tris HCl (pH 8)
    1 M
    2.5 ml
    150 mM NaCl
    1 M
    7.5 ml
    1% NP-40
    20%
    2.5 ml
    0.5% sodium deoxycholate
    10%
    2.5 ml
    0.1% SDS
    10%
    0.5 ml
  4. At time of use, add the following into 10 ml of NL buffer
                               
    Stocks              
    10 ml 1x solution
    PI cocktail
    40x
    250 μl
    10% glycerol
    -
    1 ml

    Note: DTT can be added if further delicate experiments such as IP and ChIP are needed; current recipe without DTT is fine for Western blotting.
  5. Brilliant Blue R staining solution and destaining solution
    For staining solution, dissolve 1 g of Brilliant Blue powder in 1 L of 50% Methanol/10% AceticAcid/40% H2O (all [v/v]) solution. Stir until dissolved and (optional) filter through Whatman filter paper.
    For destaining, make a 10% AceticAcid/15% Methanol/75% H2O solution.
    Procedures: Place the gel containing the proteins of interest in a plastic container and cover with fresh staining solution. Shake it for 1 h at room temperature (or overnight). Remove the staining solution and add destaining solution. Put three sheets of fine-grade tissue paper in the container. Shake it until gel is fully destained. Constantly replace the solution and tissue paper.

Acknowledgments

This protocol is based on that from Dr. Richard Patten at Abcam (see Reference 1), and was modified and utilized in a publication by Huang et al. (2012).

References

  1. Patten R. Procedure for separating nuclear, membrane, and cytoplasmic cell fractions using centrifugation methods.
  2. Huang, Z., Cheng, Y., Chiu, P. M., Cheung, F. M., Nicholls, J. M., Kwong, D. L., Lee, A. W., Zabarovsky, E. R., Stanbridge, E. J., Lung, H. L. and Lung, M. L. (2012). Tumor suppressor Alpha B-crystallin (CRYAB) associates with the cadherin/catenin adherens junction and impairs NPC progression-associated properties. Oncogene 31(32): 3709-3720.

简介

亚细胞定位对于蛋白质的正常功能是至关重要的。 亚细胞定位的失调可能导致病理结果并导致诸如癌症的疾病。 免疫荧光染色和亚细胞分级分离可用于确定蛋白质的定位。 在这里我们讨论使用离心机和超速离心机分离培养的人类细胞系的核,细胞溶质和膜部分的协议。 膜级分含有质膜和ER-高尔基体膜,但没有线粒体或核结构。 可以使用蛋白质印迹法进一步分析级分。 该协议基于来自Abcam的Richard Patten博士,并且在Huang等人的出版物中被修改和利用。 (2012)。

关键字:细胞核, 细胞溶质的, 膜, 离心, 分馏

材料和试剂

  1. 蔗糖
  2. HEPES
  3. 氯化钾(KCl)
  4. 氯化镁(MgCl 2)
  5. 乙二胺四乙酸(EDTA)
  6. 乙二醇四乙酸(EGTA)
  7. 二硫苏糖醇(DTT)
  8. Tris(Affymetrix-USB,目录号:75825)
  9. 氯化钠(NaCl)(Sigma-Aldrich,目录号:13565)
  10. Nonidet P40替代品(NP40)
  11. 脱氧胆酸钠
  12. 甘油
  13. 十二烷基硫酸钠(SDS)
  14. 蛋白酶抑制剂(PI)混合物(F.Hoffmann-La Roche,目录号:11836145001)
  15. 甲醇
  16. 乙酸
  17. Brilliant Blue R(Affymetrix,目录号:32826)
  18. 磷酸盐缓冲盐水(PBS)
  19. 组蛋白H3抗体(Cell Signaling Technology,目录号:9715)
  20. α-微管蛋白抗体(GeneTex,目录号:GTX108784)
  21. 细胞刮刀(BD Biosciences,Falcon,目录号:353086)
  22. 亚细胞分级缓冲液(SF缓冲液)(参见配方)
  23. 核裂解缓冲液(NL缓冲液)(参见配方)
  24. Brilliant Blue R染色溶液和脱色溶液(参见配方)

设备

  1. 4℃微量离心机(Eppendorf,目录号:5415R);
  2. 超速离心机(Beckman Coulter,型号:Optima TLX)
  3. 可选:Sonicator(Sonics,型号:VC505)
  4. Whatman滤纸
  5. 37℃孵育器
  6. 1.5 ml Eppendorf微管
  7. 管辊(Maplelab-scientific,型号:MTR-1D)

程序

注意:将样品在冰上保持在4°C! 所有缓冲液在使用时必须是冰冷的。 除非另有说明,所有离心均在Eppendorf Microcentrifuge中进行。

  1. 在100mm培养板上培养细胞,直到在5%CO 2提供的37℃培养箱中75%汇合。 对于初学者,建议良好研究的HEK293及其衍生物易于维护和异位蛋白表达。
    注意:这是为贴壁细胞。 悬浮细胞可能需要在裂解前离心。
  2. 用冰冷的PBS洗涤两次,立即添加500微升每100毫米板的SF缓冲液,并放在冰上,使用细胞刮刀收集裂解液,并转移到1.5毫升的Eppendorf管。 如果收集多个样品,一次处理一个样品。
  3. 在4℃下搅拌裂解物30分钟,在管辊上以约30-50rpm搅拌
  4. 在4℃下以720×g离心5分钟。 小心转移上清液到一个新的1.5毫升管以备将来使用。 保持沉淀物下一步。
  5. 用500μlSF缓冲液洗涤沉淀,用移液管分散沉淀。
  6. 在4℃下以720×g离心沉淀10分钟。
  7. 除去上清液,并在NL缓冲液中重悬沉淀。 搅拌并在4℃孵育15分钟。
    任选:在冰上超声处理沉淀(2×3秒超声处理,通过3秒静息,在30%全幅度功率下分离,在冰上! 这是包括核膜的核部分。
  8. 将来自步骤4的上清液在4℃以10,000×g离心10分钟。
  9. 小心转移上清液到新的1.5 ml管。 这是胞质和膜部分。
  10. 在超速离心机中离心步骤9的细胞溶质和膜级分。 在4℃下以100,000xg超速离心1小时。 小心转移上清液到新的1.5 ml管。 这是胞质部分。
  11. 用500μlSF缓冲液洗涤沉淀,并通过吸移重悬。
  12. 在4℃下将所述沉淀以100,000xg超速离心1小时。
  13. 除去上清液并将沉淀重悬在NL缓冲液中。
    任选:在冰上超声处理沉淀(与步骤7中的核级分相同的设置)。这是膜部分。
  14. 可以使用Western印迹的内部负荷控制来确保每个级分不会交叉污染其他级分;但是也可以在样品之间确定相对量以确保相等的负载。例如,α-微管蛋白用于胞质部分;组蛋白H3用于核部分;亮蓝R用于膜部分的染色。使用额外的凝胶装载控制。在将蛋白质转移到PVDF膜上之后探测α-微管蛋白和组蛋白H3。 Brilliant Blue R染色可直接用于SDS-PAGE凝胶(图1)。
    注意:Brilliant Blue R在这里用于监测不同样品之间蛋白质负载的相对量,但不能显示交叉污染。膜蛋白例如EGF受体和整联蛋白可以用于证实膜和其它级分之间的交叉污染。然而,这些膜蛋白的回收可能是一个问题,这可能是假阳性的交叉污染。


    图1.核,细胞质和膜级分的Western印迹 具有内部对照。α-微管蛋白和组蛋白H3用于 细胞质和核级分;辉煌蓝R 染色用于膜级分

食谱

  1. 亚细胞分级缓冲液(SF缓冲液)

    股票                   
    50 ml 1x解决方案      
    250 mM蔗糖 -
    4.28克
    20mM HEPES(pH7.4) 1 M
    1 ml
    10 mM KCl
    -
    0.0373克
    1.5mM MgCl 2·h/v 1 M
    75微升
    1mM EDTA
    0.5 M
    100微升
    1 mM EGTA
    0.5 M
    100微升
  2. 使用时,将以下物质加入10ml SF缓冲液
                                                   
    股票
    10毫升1x解决方案                  
    1 mM DTT
    1 M
    10微升
    PI鸡尾酒
    40x(在2ml dd H 2 O中溶解1片) 250微升
  3. 核裂解缓冲液(NL缓冲液)

    股票                        
    50 ml 1x解决方案           
    50mM Tris HCl(pH8)
    1 M
    2.5 ml
    150mM NaCl 1 M
    7.5 ml
    1%NP-40
    20%
    2.5 ml
    0.5%脱氧胆酸钠 10%
    2.5 ml
    0.1%SDS
    10%
    0.5ml
  4. 使用时,将以下物质加入10ml NL缓冲液
                               
    股票               
    10ml 1x溶液
    PI鸡尾酒
    40x
    250微升
    10%甘油 -
    1 ml

    注意:如果需要进一步细致的实验,如IP和ChIP,可以添加DTT; 目前没有DTT的食谱适用于Western印迹。
  5. Brilliant Blue R染色溶液和脱色溶液 对于染色溶液,将1g亮蓝色粉末溶解在1L的50%甲醇/10%乙酸/40%H 2 O(全[v/v])溶液中。 搅拌直至溶解并(可选)通过Whatman滤纸过滤 为了脱色,制备10%乙酸/15%甲醇/75%H 2 O溶液。
    程序:将含有感兴趣的蛋白质的凝胶置于塑料容器中,并盖上新鲜的染色溶液。 在室温下摇动1小时(或过夜)。 取出染色溶液并加入脱色溶液。 将三片细级棉纸放入容器中。摇动,直到凝胶完全脱色。不断更换溶液和薄纸。

致谢

该协议基于来自Abcam的Richard Patten博士(参见参考文献1),并在Huang等人(2012)的出版物中进行了修改和使用。

参考文献

  1. Patten R. 使用离心方法分离核,细胞膜和细胞质细胞级分的程序。
  2. Huang,Z.,Cheng,Y.,Chiu,P.M.,Cheung,F.M.,Nicholls,J.M.,Kwong,D.L.,Lee,A.W.,Zabarovsky,E.R.,Stanbridge,E.J.Lung,H.L.and Lung, 肿瘤抑制基因Alpha B晶状体蛋白(CRYAB)与钙粘着蛋白/连环蛋白粘附连接相关,并损害NPC进展相关属性。 Oncogene 31(32):3709-
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yu, Z., Huang, Z. and Lung, M. L. (2013). Subcellular Fractionation of Cultured Human Cell Lines. Bio-protocol 3(9): e754. DOI: 10.21769/BioProtoc.754.
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Md Wasim Khan
University of Illinois at Chicago
There is no homogenization step in the protocol. Will the cells just swell and burst due to the sucrose?
2020/1/29 9:05:28 回复
Pia Giovannelli
University of Campania "L.Vanvitelli"
This protocol is very simple to reproduce. I isolated only the nuclear and cytoplasmic fractions, not membrane. I added two more washes with SF buffer before usinf the NL buffer because into the nuclear fraction I had too much tubulin.
2018/11/30 2:56:12 回复
Daniel Miller
Daniel Miller
For suspension cells, how many cells would you start with?

How much NL buffer would you resuspend the cells with--or is it dependent on pellet size?

Thanks!
2018/8/6 7:39:03 回复
Yu-Jou Tsai
National Cheng Kung University
Thank you for replying
Since you only use 500ul for re-suspending and then ultracentrifuge, I'd like to ask what kind of ultracentrifuge tube you use?
Thank you!
2018/7/5 3:28:28 回复
Yu-Jou Tsai
National Cheng Kung University
Which kind of rotor do you use? SW28 or SW40ti ?
Thank you!
2018/7/4 22:35:15 回复
Maria Lung
Clinical Oncology Department, The Univerisity of Hong Kong, Hong Kong SAR

Dear Yu-Jou,

we use SW41 Ti.

regards

2018/7/4 23:14:14 回复


Angela Russo
UIC
Hello,
I would like to know the pH of EDTA and EGTA stocks for the preparation of the fractionation buffer.
Thanks,
Angela
2015/3/18 12:29:15 回复
Maria Lung
Clinical Oncology Department, The Univerisity of Hong Kong, Hong Kong SAR

Dear Angela,

we use 0.5M pH8 (adjusted using NaOH) EDTA and EGTA stocks for the preparation.

yours
Valen Z. YU

2015/3/27 2:07:45 回复