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Membrane Flotation Assay
膜漂浮分析   

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参见作者原研究论文

本实验方案简略版
PLOS Pathogens
Apr 2013

Abstract

Many postitive-stranded RNA viruses, such as Hepatitis C virus (HCV), highjack cellular membranes, including the Golgi, ER, mitchondria, lipid droplets, and utilize them for replication of their RNA genome or assembly of new virions. By investigating how viral proteins associate with cellular membranes we will better understand the roles of cellular membranes in the viral life cycle. Our lab has focused specifically on the role of lipid droplets and lipid-rich membranes in the life cycle of HCV. To analyze the role of lipid-rich membranes in HCV RNA replication, we utilized a membrane flotation assay based on an 10-20-30% iodixanol density gradient developed by Yeaman et al. (2001). This gradient results in a linear increase in density over almost the entire length of the gradient, and membrane particles are separated in the gradient based on their buoyant characteristics. To preserve membranes in the lysate, cells are broken mechanically in a buffer lacking detergent. The cell lysate is loaded on the bottom of the gradient, overlaid with the gradient, and membranes float up as the iodixanol gradient self-generates. The lipid content of membranes and the concentration of associated proteins will determine the separation of different membranes within the gradient. After centrifugation, fractions can be sampled from the top of the gradient and analyzed using standard SDS-PAGE and western blot analysis for proteins of interest.

Materials and Reagents

  1. Huh7.5 cells
  2. Huh7.5 cells containing replicating HCV Replicon RNA (= Replicon Cells) (Vogt et al., 2013)
  3. Dulbecco’s Modified Eagle’s Medium (with 4.5 g/L glucose, L-glutamine & Sodium pyruvate) (DMEM) (Corning Cellgro, catalog number: 10-013-CV )
  4. Fetal Bovine Serum (FBS) (Benchmark, catalog number: 100-106 )
  5. Penicillin/Streptomicyn Solution 100x (Pen/Strep) (Corning Cellgro, catalog number: 30-002-Cl )
  6. L-Glutamine (Corning Cellgro, catalog number: 25-005-CI )
  7. G418 sulfate (Corning Cellgro, catalog number: 25-052-CI )
  8. Dulbecco’s Phosphate Buffered Saline without Ca2+ and Mg2+ (PBS) (Corning Cellgro, catalog number: 21-031-CV )
  9. Trypsin/EDTA (Corning Cellgro, catalog number: 25-052-CI)
  10. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 ) (use as 1:100 dilution)
  11. Trypan Blue Stain (Gibco, catalog number: 15250-061 )
  12. BioRad Protein Assay (Bio-Rad Laboratories, catalog number: 500-0006 )
  13. Brilliant Blue G-250 (Thermo Fisher Scientific, catalog number: 100-25 )
  14. Iodixanol (60%) (Sigma-Aldrich, catalog number: D1566-250ML ) (kept at 4 °C for use in the experiment )
  15. PBS/Sucrose (see Recipes)
  16. 20% iodixanol (see Recipes)
  17. 10% iodixanol (see Recipes)
  18. 2x Laemmli buffer (see Recipes)

Equipment

  1. Ultra clear centrifuge tubes (Beckman Coulter, catalog number: 344059 )
  2. 37 °C 5% CO2 cell culture incubator
  3. P1000 pipette
  4. Microscope
  5. Hematocytometer
  6. Tight fitting dounce homogenizer (7 ml) (Wheaton, catalog number: 357542 )
  7. Centrifuge (Beckman Coulter, model: Allegra 6R )
  8. Spectrophotometer
  9. Ultracentrifuge (Beckman Coulter, model: Optima L-80 XP with SW41T rotor)
  10. Protein electrophoresis apparatus
  11. Western blot apparatus

Procedure

  1. Huh7.5 or HCV replicon cells are grown in DMEM supplemented with 10% FBS, 1x Pen/Strep, 1x Glutamine for 4-5 days from 20% to a 80% final confluency in T175 flasks (will need 5 flasks per cell type). The medium for HCV replicon cells is additionally supplemented with 800 μg/ml of G418.
  2. Cells are harvested by washing once with 10 ml of PBS (add PBS to flask, agitate by hand 2-3 times and aspirate PBS off), then trypsinized with 4 ml of trypsin per T175 flask (incubate cells with trypsin at 37 °C for 3-5 min, take off cells with 6 ml of PBS by washing the walls of the flask and transferring to a 50 ml cornical tube). Cells are spun down for 5 min at 400 x g, the supernatant is aspirated off, and cells are washed once with 30 ml PBS by resuspending cells in PBS and spun down again for 5 min at 400 x g, then resuspended in 10 ml PBS, and counted in a hematocytometer. A total of 3 x 107 cells for each sample are resuspended in 3.5 ml pre-chilled PBS/Sucrose plus protease inhibitor cocktail (diluted 1:100) and immediately stored on ice.
  3. The cells are lysed with 200 passages in a tight-fitting dounce homogenizer, while keeping the homogenizer on ice.
    Note: Be careful not to lift the pestle above the surface of the lysate to minimized creation of air bubbles.
  4. Check that cells are over 90% lysed by mixing 10 μl of the lysate with 10 μl Trypan Blue stain, then checking the sample in the microscope. Dead cells will be stained blue. If not enough cells are lysed, lyse cells with additional passages in the dounce homogenizer, and check every 50 passages that cell lysis was successful.
  5. The cell lysate is spun at 2,500 x g for 10 min at 4 °C to pellet cellular debris and nuclei.
  6. Transfer the supernatant (referred to as crude lysate) to a fresh tube and keep on ice.
  7. Measure total protein concentration in the crude lysate using Bio-Rad Protein Assay according to the manufacturer’s protocol.
  8. Take 5 mg of protein for each sample and add PBS/Sucrose to a total volume of 2 ml. Freeze the rest of the crude lysate mixed 1:1 with 2x Laemmli buffer as a control.
  9. Mix 2 ml of sample 1:1 with 2 ml of 60% iodixanol (pre-chilled at 4 °C) to get 4 ml of sample at a final concentration of 30% iodixanol.
  10. Put the 4 ml of 30% iodixanol/lysate mixture on the bottom of a centrifuge tube. Overlay carefully with 4 ml of 20% iodixanol (pre-chilled), then overlay with 4 ml of 10% iodixanol (pre-chilled). Then fill the tube with 10% until the level reaches 1 mm below the rim of the centrifuge tube. (Overlay with a P1000 pipette by cutting off 1 cm of the pipette tips, then pipette slowly 1 ml at the time right into the middle of the centrifuge tube right on top of the gradient level, to minimize mixing.) Make sure the tubes are balanced well for centrifugation, add carefully 10% Iodixanol on top to balance tubes if necessary.
  11. Spin at 209,000 x g (35,000 rpm) overnight (16 h) at 4 °C in an SW41T rotor.
  12. Next day: Collect 22 fractions (500 μl) each from top to bottom. Use cut off pipette tips of P1000 and carefully take off 500 μl from the middle by just touching the pipette tip to the surface of the gradient, then lowering the tip slowly to stay right at the surface of the gradient while taking off the sample very slowly. The increased width of the pipette tip decreases a whirlpool effect and minimizes mixing of the gradient layers.
  13. Mix fractions 1:1 with 2x Laemmli buffer, and freeze, preferable in aliquots, at -80 °C.
  14. Samples can then be analyzed via SDS-PAGE and Western blots [see results in Vogt et al. (2013)].

Notes

  1. This protocol could be applied to other cell lines as well. The distribution among the fractions at the end may vary depending on the cell line. Cells lines with a very low lipid content may not work as well as the Huh7.5, however, the general protocol could be applied (and could be optimized a bit) to most cell lines since they all contain cellular membranes.
  2. For detailed information on how to determine lipid content for whole cell lysate, readers are recommended to read the review article by Camus et al. (2013).

Recipes

  1. PBS/Sucrose
    Dissolve 8.55 g sucrose in 100 ml of PBS to a final concentration of 0.25 M sucrose in PBS
    Kept at 4 °C
  2. 20% Iodixanol
    10 ml of Iodixanol (60%)
    20 ml of PBS/Sucrose
    Kept at 4 °C
  3. 10% Iodixanol
    5 ml of Iodixanol (60%)
    25 ml of PBS/Sucrose
    Kept at 4 °C
  4. 2x Laemmli buffer
    125 mM tris-HCl (pH 6.8)
    20% glycerol
    2.5% SDS
    2.5 mg/100 ml Brilliant Blue G-250
    Mix fresh before use: 950 μl of 2x Laemmli buffer with 50 μl β-mercaptoethanol

Acknowledgments

This work was supported by funds from the Gladstone Institutes, and the National Institutes of Health (R056 AI069090 (MO), and P30 DK026743 (MO) (University of California – San Francisco Liver Center). We gratefully acknowledge support through the Training Grant (T32 DK060414) from the US National Institute of Health to DAV.

References

  1. Camus, G., Vogt, D. A., Kondratowicz, A. S. and Ott, M. (2013). Lipid droplets and viral infections. Methods Cell Biol 116: 167-190.
  2. Vogt, D. A., Camus, G., Herker, E., Webster, B. R., Tsou, C. L., Greene, W. C., Yen, T. S. and Ott, M. (2013). Lipid droplet-binding protein TIP47 regulates hepatitis C Virus RNA replication through interaction with the viral NS5A protein. PLoS Pathog 9(4): e1003302.
  3. Yeaman, C., Grindstaff, K. K., Wright, J. R. and Nelson, W. J. (2001). Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells. J Cell Biol 155(4): 593-604.

简介

许多假性绞线的RNA病毒,例如丙型肝炎病毒(HCV),高压细胞膜,包括高尔基体,ER,马钱子树,脂滴,并利用它们复制其RNA基因组或组装新的病毒粒子。通过调查病毒蛋白如何与细胞膜结合,我们将更好地了解细胞膜在病毒生命周期中的作用。我们的实验室特别关注脂质滴和富含脂质的膜在HCV的生命周期中的作用。为了分析富含脂质的膜在HCV RNA复制中的作用,我们使用基于由Yeaman等人(2001)开发的10-20-30%碘克沙醇密度梯度的膜浮选测定。该梯度导致在梯度的几乎整个长度上密度的线性增加,并且膜颗粒基于其浮力特性在梯度中分离。为了保存裂解物中的膜,在缺乏洗涤剂的缓冲液中机械破碎细胞。将细胞裂解物加载在梯度的底部,用梯度覆盖,并且膜随着碘克沙醇梯度自我产生而浮起。膜的脂质含量和相关蛋白的浓度将决定梯度内不同膜的分离。离心后,可以从梯度的顶部取样级分,并使用标准SDS-PAGE和蛋白质印迹分析来分析感兴趣的蛋白质。

材料和试剂

  1. Huh7.5细胞
  2. 含有复制型HCV复制子RNA(=复制子细胞)的Huh7.5细胞(Vogt等人,2013)
  3. Dulbecco改良的Eagle培养基(含有4.5g/L葡萄糖,L-谷氨酰胺和丙酮酸钠)(DMEM)(Corning Cellgro,目录号:10-013-CV)
  4. 胎牛血清(FBS)(Benchmark,目录号:100-106)
  5. 青霉素/链霉素溶液100x(Pen/Strep)(Corning Cellgro,目录号:30-002-Cl)
  6. L-谷氨酰胺(Corning Cellgro,目录号:25-005-CI)
  7. G418硫酸盐(Corning Cellgro,目录号:25-052-CI)
  8. 没有Ca 2+和Mg 2+ 2+(PBS)(Corning Cellgro,目录号:21-031-CV)的Dulbecco's磷酸盐缓冲盐水
  9. 胰蛋白酶/EDTA(Corning Cellgro,目录号:25-052-CI)
  10. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)(以1:100稀释度使用)
  11. 台盼蓝染料(Gibco,目录号:15250-061)
  12. BioRad蛋白测定(Bio-Rad Laboratories,目录号:500-0006)
  13. Brilliant Blue G-250(Thermo Fisher Scientific,目录号:100-25)
  14. 碘克沙醇(60%)(Sigma-Aldrich,目录号:D1566-250ML)(保持在4℃用于实验中)
  15. PBS /蔗糖(见配方)
  16. 20%iodixanol(见配方)
  17. 10%iodixanol(见配方)
  18. 2x Laemmli缓冲区(参见配方)

设备

  1. 超清离心管(Beckman Coulter,目录号:344059)
  2. 37℃5%CO 2细胞培养孵育器
  3. P1000移液器
  4. 显微镜
  5. 血细胞计数器
  6. 紧配合杜松匀浆器(7ml)(Wheaton,目录号:357542)
  7. 离心机(Beckman Coulter,型号:Allegra 6R)
  8. 分光光度计
  9. 超速离心机(Beckman Coulter,型号:Optima L-80 XP,带SW41T转子)
  10. 蛋白电泳仪
  11. Western印迹仪

程序

  1. Huh7.5或HCV复制子细胞在补充有10%FBS,1x Pen/Strep,1×谷氨酰胺的DMEM中生长4-5天,在T175烧瓶中从20%至80%最终融合(每种细胞类型需要5个烧瓶) 。用于HCV复制子细胞的培养基另外补充有800μg/ml G418
  2. 通过用10ml PBS(加入PBS至烧瓶,手动搅拌2-3次并抽出PBS)洗涤一次,然后用每个T175烧瓶中的4ml胰蛋白酶胰蛋白酶消化细胞(在37℃下用胰蛋白酶孵育细胞,用于3-5分钟,通过洗涤烧瓶壁并转移到50ml锥形管中,用6ml PBS取出细胞)。将细胞以400×g离心5分钟,吸出上清液,通过将细胞重悬浮于PBS中,用30ml PBS洗涤细胞一次,并在400rpm下再次离心5分钟, xg,然后重悬于10ml PBS中,并在血细胞计数器中计数。将每个样品的总共3×10 7个细胞重悬于3.5ml预冷的PBS /蔗糖加蛋白酶抑制剂混合物(1:100稀释)中,立即储存在冰上。
  3. 在紧密配合的dounce匀浆器中将细胞裂解200次,同时将匀浆器保持在冰上。
    注意:小心不要将杵提起在裂解液表面以上,以尽量减少气泡的产生。
  4. 通过混合10微升的溶胞产物与10微升台盼蓝染色,然后检查样品在显微镜检查细胞超过90%裂解。死细胞会染成蓝色。如果没有足够的细胞裂解,在dounce匀浆器中额外传代裂解细胞,并检查每50次传代细胞裂解是成功的。
  5. 将细胞裂解物在4℃下以2,500xg离心10分钟以沉淀细胞碎片和细胞核。
  6. 将上清液(称为粗裂解物)转移到新管中并保持在冰上。
  7. 使用Bio-Rad蛋白测定根据制造商的方案测量粗裂解物中的总蛋白浓度
  8. 每个样品取5毫克蛋白质,加入PBS /蔗糖至总体积为2毫升。将2×Laemmli缓冲液以1:1混合的剩余粗裂解物冻结作为对照
  9. 将2ml样品与2ml 60%碘克沙醇(在4℃预冷)1:1混合,得到4ml终浓度为30%碘克沙醇的样品。
  10. 将4毫升30%iodixanol /裂解液混合物放在离心管的底部。小心地用4ml 20%碘克沙醇(预冷冻)覆盖,然后用4ml 10%碘克沙醇(预冷冻)覆盖。然后用10%填充管,直到水平达到离心管边缘以下1mm。 (用P1000移液管通过切下1cm的移液管吸头覆盖,然后慢慢地移取1ml,立即在离心管中间的梯度水平的顶部缓慢移动,以使混合最小化。)确保管平衡孔离心,如果需要,小心地添加10%的碘克沙醇到平衡管
  11. 在SW41T转子中于4℃下以209,000×g(35,000rpm)旋转过夜(16小时)。
  12. 第二天:从顶部到底部收集22个级分(500μl)。使用切断P1000的移液器吸头,并从中间小心取出500微升,只是触摸移液器的尖端到梯度的表面,然后缓慢下降尖端保持在梯度的表面,同时脱离样品非常缓慢。移液管尖端的增加的宽度减小了漩涡效应并且使梯度层的混合最小化。
  13. 混合级分1:1与2x Laemmli缓冲液,并冷冻,优选等分,在-80℃。
  14. 然后可以通过SDS-PAGE和Western印迹分析样品[参见Vogt等人(2013)的结果]。

笔记

  1. 该协议也可以应用于其他细胞系。 末端部分之间的分布可以根据细胞系而变化。 具有非常低脂质含量的细胞系可能不如Huh7.5那样工作,然而,一般方案可以应用于(并且可以优化一点)大多数细胞系,因为它们都含有细胞膜。
  2. 关于如何确定全细胞裂解物的脂质含量的详细信息,读者推荐阅读Camus等人(2013)的综述文章。

食谱

  1. PBS /蔗糖
    将8.55g蔗糖溶解在100ml PBS中,使其最终浓度为0.25M蔗糖的PBS溶液
    保持在4°C
  2. 20%碘克沙醇
    10ml的碘克沙醇(60%)
    20ml PBS /蔗糖 保持在4°C
  3. 10%碘克沙醇
    5ml的碘克沙醇(60%)
    25ml PBS /蔗糖 保持在4°C
  4. 2x Laemmli缓冲区
    125mM tris-HCl(pH 6.8)
    20%甘油 2.5%SDS
    2.5mg/100ml Brilliant Blue G-250 使用前混合新鲜:950μl2x Laemmli缓冲液和50μlβ-巯基乙醇

致谢

这项工作得到Gladstone研究所和国家卫生研究院(R056 AI069090(MO)和P30 DK026743(MO)(加利福尼亚大学 - 旧金山肝脏中心)的资金支持,我们衷心感谢通过培训资助 T32 DK060414)从美国国家卫生研究所DAV。

参考文献

  1. Camus,G.,Vogt,D.A.,Kondratowicz,A.S。和Ott,M。(2013)。 脂滴和病毒感染 方法细胞生物学 116 :167-190。
  2. Vogt,D.A.,Camus,G.,Herker,E.,Webster,B.R.,Tsou,C.L.,Greene,W.C.,Yen,T.S.and Ott, 脂质小滴结合蛋白TIP47通过与病毒NS5A蛋白相互作用来调节丙型肝炎病毒RNA复制。/a> PLoS Pathog 9(4):e1003302。
  3. Yeaman,C.,Grindstaff,K.K.,Wright,J.R.and Nelson,W.J。(2001)。 跨高尔基网络和质膜上的Sec6/8复合物调节哺乳动物细胞中胞吐作用的晚期。 J Cell Biol 155(4):593-604
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vogt, D. A. and Ott, M. (2015). Membrane Flotation Assay. Bio-protocol 5(7): e1435. DOI: 10.21769/BioProtoc.1435.
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