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Enrichment of Golgi membranes from HeLa cells by sucrose gradient ultracentrifugation
通过蔗糖梯度超速离心法从HeLa细胞中富集高尔基体膜   

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Abstract

This is a protocol to extract intact Golgi Membranes from HeLa cells using sucrose gradient centrifugation. This extraction is very useful for several applications including pull-down of Golgi membrane proteins, electron microscopy and reconstitution of protein transport into an isolated system. Protocol adapted from Balch et al. (1984).

Keywords: Golgi membranes(高尔基体膜), HeLa cells(HeLa细胞), Sucrose gradient cenrifugation(蔗糖梯度cenrifugation)

Materials and Reagents

  1. HeLa cells (ATTC, Wesel, Germany)
  2. PBS
  3. 1 M Tris (pH 7.4)
  4. 100 mM EDTA
  5. Trypan Blue
  6. Protease inhibitor cocktail tablets (Roche, catalog number: 11836153001 )
  7. Breaking buffer (BB) (see Recipes)
  8. 29% (w/w) sucrose (see Recipes)
  9. 35% (w/w) sucrose (see Recipes)
  10. 62% (w/w) sucrose (see Recipes)

Equipment

  1. Cell scrapers
  2. Cell homogenizer (EMBL cell cracker) (EMBLEM Technology Transfer, Heidelberg)
  3. Cell culture microscope
  4. Ultracentrifuge (Beckman Coulter, model: Optima L-100K or equivalent)
  5. Refractometer
  6. SW40Ti rotor
  7. Centrifuge tubes
  8. 1 ml syringe with 20/21 G needle

Procedure

  1. Remove medium and wash cells 3x with PBS and 1x with Breaking buffer (BB).
  2. Harvest the cells by scraping and pellet the cells (for instance at 300 x g, 5 min).
  3. Wash pellet 2x in PBS centrifuge cells at 300 x g, 5 min.
  4. Wash 1x in ice-cold BB.
  5. Dilute the pellet 1:5 in ice-cold BB.
  6. Homogenize pellet with an EMBL cell cracker 20x on ice.
    Note: Make sure there are no air bubbles during the homogenization.
  7. Mix a few μl of homogenate with a trypan blue solution on a glass slide and cover it with a coverslip. Check homogenization by microscope.
    Note: Plasma membrane should not be intact anymore. Cell nuclei should stain blue with Trypan Blue. There should be a lot of membrane fragments and particles in the homogenate, but the nucleus should stay intact.
  8. Mix the homogenate with 62% sucrose
    1. 2 ml homogenate
    2. 1.83 ml of 62% ice-cold sucrose
    3. 41.7 μl of 100 mM EDTA (pH 7.4)
    Check the sucrose concentration to 37% +/- 0.5% with a refractometer.
    Sucrose gradients: solutions are w/w%.
    Check pH of the solutions after dissolving the sucrose.
  9. Run gradient
    1. 4 ml homogenate (in 37% sucrose)
    2. 4.5 ml 35% sucrose
    3. 3.5 ml 29% sucrose (to the top)
    Note: Homogenate at the bottom, then add 35% sucrose, then add 29% sucrose.
  10. Centrifugation: SW 40 Ti Rotor, centrifuge for 1.5 h at max speed (x g) at 4 °C.



  11. Pull the Golgi band in 0.4 ml using a 1 ml syringe with 20/21 G needle (the Golgi band is located at the 35%/29% sucrose interphase).
  12. Measure protein concentration and the functional Golgi membranes can now be snap frozen in liquid N2 and stored at -80 °C.

Notes

  1. Avoid salts/ions in the homogenate as it may aggregate the organelles.
  2. Addition of high amount of sucrose affects the pH.
  3. Don’t homogenize too much in step 6 as organelles can break
    1. Proteases can leak out of the lysosomes.
    2. Broken organelles can reseal with other broken organelles.
    3. DNA can be released from nuclei which makes the sample sticky.
  4. The isolated Golgi membranes are in a buffer containing about 30% of sucrose. Therefore, if Golgi membranes need to be pelleted for further analysis, the sucrose needs to be diluted out by addition of 3 volumes of an appropriate buffer such as PBS.

Recipes

  1. Breaking buffer (BB)
    250 mM Sucrose
    10 mM Tris (pH 7.4) 
    Add protease inhibitor cocktail tablets
  2. 29% (w/w) sucrose
    65.08 g sucrose/200 ml
    10 mM Tris (pH 7.4)
  3. 35% (w/w) sucrose
    80.60 g sucrose/200 ml
    10 mM Tris (pH 7.4)
  4. 62% (w/w) sucrose
    161 g sucrose/200 ml
    10 mM Tris (pH 7.4)
    Note: Check all sucrose solutions with refractometer index and % of sucrose.

Acknowledgments

The protocol was adapted from the original version published by Balch et al. (1984).

References

  1. Balch, W. E., Dunphy, W. G., Braell, W. A. and Rothman, J. E. (1984). Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell 39(2): 405-416.
  2. von Blume, J., Alleaume, A.-M., Kienzle, C., Carreras-Sureda, A., Valverde, M. and Malhotra, V. (2012). Cab45 is required for Ca2+-dependent secretory cargo sorting at the trans-Golgi network. J Cell Biol 199(7): 1057-1066.

简介

这是一种使用蔗糖梯度离心从HeLa细胞提取完整高尔基膜的方案。 这种提取对于包括高尔基体膜蛋白的下拉,电子显微镜和将蛋白质转运到分离系统中的重新应用是非常有用的。 Balch et al。(1984)。

关键字:高尔基体膜, HeLa细胞, 蔗糖梯度cenrifugation

材料和试剂

  1. HeLa细胞(ATTC,Wesel,Germany)
  2. PBS
  3. 1M Tris(pH7.4)
  4. 100 mM EDTA
  5. 台盼蓝
  6. 蛋白酶抑制剂混合物片剂(Roche,目录号:11836153001)
  7. 断开缓冲区(BB)(参见配方)
  8. 29%(w/w)蔗糖(见Recipes)
  9. 35%(w/w)蔗糖(参见配方)
  10. 62%(w/w)蔗糖(见Recipes)

设备

  1. 细胞刮刀
  2. 细胞匀浆器(EMBL细胞裂解器)(EMBLEM Technology Transfer,Heidelberg)
  3. 细胞培养显微镜
  4. 超速离心机(Beckman Coulter,型号:Optima L-100K或等同物)
  5. 折射计
  6. SW40Ti转子
  7. 离心管
  8. 1 ml注射器,带20/21 G针

程序

  1. 取出培养基,用PBS洗涤细胞3次,用破碎缓冲液(BB)洗涤1次。
  2. 通过刮擦和沉淀细胞来收获细胞(例如以300×g/5分钟,5分钟)。
  3. 在300×g /分钟,5分钟的PBS离心细胞中洗涤沉淀2次
  4. 在冰冷的BB中洗涤1次。
  5. 在冰冷的BB中将沉淀物1:5稀释
  6. 在冰上用EMBL细胞破碎器20x匀浆沉淀 注意:确保均质化过程中没有气泡。
  7. 混合几个匀浆与台盼蓝溶液在载玻片上,盖上盖玻片。 用显微镜检查匀浆 注意:质膜不应该是完整的。 细胞核应用台盼蓝染蓝色。 在匀浆中应该有很多膜片段和颗粒,但是核应该保持完整。
  8. 将匀浆与62%蔗糖混合
    1. 2ml匀浆
    2. 1.83ml 62%冰冷的蔗糖
    3. 41.7μl的100mM EDTA(pH7.4)
    用折射计检查蔗糖浓度为37%+/- 0.5% 蔗糖梯度:溶液w/w% 检查溶解蔗糖后溶液的pH值
  9. 运行渐变
    1. 4ml匀浆(在37%蔗糖中)
    2. 4.5ml 35%蔗糖
    3. 3.5ml 29%蔗糖(至顶部)
    注意:在底部均化,然后加入35%蔗糖,然后加入29%蔗糖。
  10. 离心:SW 40 Ti Rotor,在4℃以最大速度离心1.5小时( x g )。



  11. 使用1ml注射器用20/21G针头(高尔基带位于35%/29%蔗糖相间)将0.4ml中的高尔基体带拉出。
  12. 测量蛋白质浓度并且功能性高尔基体膜现在可以在液体N 2中快速冷冻并储存在-80℃。

笔记

  1. 避免盐/离子在匀浆中,因为它可能聚集细胞器
  2. 加入大量蔗糖会影响pH值
  3. 不要在步骤6中过多匀浆,因为细胞器可能破坏
    1. 蛋白酶可以从溶酶体中漏出。
    2. 破碎的细胞器可以与其他破碎的细胞器重新密封。
    3. DNA可以从细胞核释放,使样品粘性
  4. 分离的高尔基膜在含有约30%蔗糖的缓冲液中。 因此,如果高尔基体膜需要沉淀用于进一步分析,则需要通过加入3体积的合适的缓冲液如PBS来稀释蔗糖。

食谱

  1. 断开缓冲区(BB)
    250 mM蔗糖 10 mM Tris(pH 7.4) 
    加入蛋白酶抑制剂混合物片剂
  2. 29%(w/w)蔗糖 65.08g蔗糖/200ml
    10mM Tris(pH7.4)
  3. 35%(w/w)蔗糖 80.60g蔗糖/200ml
    10mM Tris(pH7.4)
  4. 62%(w/w)蔗糖 161g蔗糖/200ml
    10mM Tris(pH7.4) 注意:检查所有蔗糖溶液与折射率指数和%的蔗糖。

致谢

该方案改编自Balch等人公布的原始版本(1984)。

参考文献

  1. Balch,W.E.,Dunphy,W.G.,Braell,W.A.and Rothman,J.E。(1984)。 通过偶联的N-乙酰葡萄糖胺的结合测量,重建高尔基体的连续区室之间的蛋白质运输 。 Cell 39(2):405-416。
  2. von Blume,J.,Alleaume,A.-M.,Kienzle,C.,Carreras-Sureda,A.,Valverde,M.and Malhotra,V.(2012)。 Cab45是Ca 2 + 依赖分泌货物分拣所需的 trans-Golgi network。 J Cell Biol 199(7):1057-1066。

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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Galen, J. v. and Blume, J. v. (2013). Enrichment of Golgi membranes from HeLa cells by sucrose gradient ultracentrifugation. Bio-protocol 3(18): e906. DOI: 10.21769/BioProtoc.906.
  2. von Blume, J., Alleaume, A.-M., Kienzle, C., Carreras-Sureda, A., Valverde, M. and Malhotra, V. (2012). Cab45 is required for Ca2+-dependent secretory cargo sorting at the trans-Golgi network. J Cell Biol 199(7): 1057-1066.

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