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Small-scale Subcellular Fractionation with Sucrose Step Gradient
蔗糖不连续密度梯度离心法进行小规模亚细胞分级分离   

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Abstract

Here, we introduce the protocol for small-scale and simple subcellular fractionation used in our recent publication (Taguchi et al., 2013), which uses homogenization by passing through needles and sucrose step-gradient.
Subcellular fractionation is a very useful technique but usually a large number of cells are required. Because we needed subcellular fractionation of transiently-transfected cells, we developed a protocol for smaller numbers of cells. Our protocol for the subcellular fractionation is based on the protocol published by de Araújo and Huber (de Araujo et al., 2007), although substantial modifications have been made according to our experiences and information from personal communications. As optimal conditions seem to vary between cell lines, we advise to further modify the protocol to optimize for individual experiments. Our method is simple but sufficient for analysis of integral membrane proteins or proteins anchored to organelles by glycosylphosphatidylinositol or other lipid anchors, e.g. prion protein. However, proteins non-covalently attached to membranes or membrane proteins of organelles seem to be more prone to dissociation from the organelles during preparation and, if these proteins are the object of study, further modifications might be necessary.
Unlike in a continuous gradient, where a protein of interest is scattered over a wide range, step-gradient fractionation is advantageous in detection of relatively small amounts of proteins from small-scale experiments, because it concentrates the protein of interest in one fraction, if an appropriate combination of sucrose concentrations is used.

Keywords: Subcellular fractionation(亚细胞分离), Sucrose step gradient(蔗糖梯度), Integral membrane protein(完整的膜蛋白), GPI-anchored protein(锚定蛋白), Prion protein(朊病毒蛋白)

Materials and Reagents

  1. Neuro2a cells (N2a)
  2. Sucrose (Sigma-Aldrich, catalog number: S9378-500G )
  3. 1 M Tris-HCl (pH 7.1)
  4. 0.5 M EDTA (Millipore, catalog number: 324503-1KG )
  5. Purified water
  6. 100 mg/ml solution of cycloheximide in DMSO (Sigma-Aldrich, catalog number: C4859-1ML )
  7. Complete protease-inhibitor cocktail (Roche Diagnostics, catalog number: 04693116001 )
  8. OptiMEM I supplemented with 10% fetal bovine serum
  9. Phosphate-buffered saline without calcium/magnesium (Ca/Mg) (Life Technologies, catalog number: 10010-023 )
  10. Deoxycholic acid (Sigma-Aldrich, catalog number: D2510-100G )
  11. Triton X-100 (Sigma-Aldrich, catalog number: 93443-100ML )
  12. Sodiumdodecyl sulfate (Sigma-Aldrich, catalog number: L6026-50G )
  13. Glycerol (Sigma-Aldrich, catalog number: G9012-500ML )
  14. Phosphate-buffered 0.5% Triton X-100 (TX100)/0.5% deoxycholate (DOC) lysis buffer (see Recipes)
  15. 5x sample buffer (see Recipes)

Equipment

  1. 6-well plate
  2. 1 ml BD Luer-LokTM disposable syringe (BD, catalog number: 309628 )
  3. 25G ultra-thin-wall needle (Terumo Medical Corporation, catalog number: NN-2525R )
  4. Cell scraper
  5. Centrifuge tube (thinwall, Ultra-ClearTM, 5 ml, 13 x 51 mm) (Beckman Coulter, catalog number: 344057 )
  6. Ultracentrifuge (Beckman Coulter, model: L8-80M )
  7. Pre-chilled swing bucket rotor (Beckman Coulter, model: SW50.1 )
  8. Portable refractometer (optional) (e.g. ATAGO, model: PAL-1 )
  9. Inverted microscope

Procedure

  1. Preparation of sucrose and homogenization buffers
    1. Decide the sucrose concentrations of the layers for the step gradient, e.g. 36%, 33% and 8.5% (Taguchi et al., 2013).
    2. The concentration of sucrose buffers is based on “percent by weight”, i.e. sucrose concentration (%) = 100 x sucrose (g)/[sucrose (g) + water (g)].
    3. Then, take necessary amounts of sucrose and dissolve it in purified water. Usually, measuring the actual concentration is not necessary as long as the amounts of sucrose and water are correct. If necessary, we confirm the actual concentration with a portable refractometer.
    4. After preparation of the sucrose solution in water, we measure the volume and add 1/250-volume of 1 M Tris-HCl (pH 7.1) to make the final concentration of 4 mM. We use Tris-HCl buffer but the buffer type should be determined based on the following procedures and the purpose of the experiments.
    5. For the homogenization buffer, the sucrose concentration of the homogenization buffer is 8.5% and, in addition to the 4 mM Tris-HCl, we add EDTA to a final concentration 2 mM, and cycloheximide to 30 µg/ml, and complete protease inhibitor cocktail with a dosage according to the manufacturer’s instruction. Cycloheximide is added to maintain the polysomes on the endoplasmic reticulum and to increase the density of the organelles for more efficient separation (de Araujo et al., 2007). 

  2. Homogenization of cells
    1. Rinse the N2a cells, 100% confluent in OptiMEM I supplemented with 10% fetal bovine serum on a well of a 6-well plate, once with pre-chilled PBS without Ca/Mg, then add 700 µl of 3 mM EDTA in PBS and incubate for a few minutes until cells can be easily detached from the bottom by pipetting.
      1. Although N2a cells can be detached with this procedure, more adhesive cells might require mechanical detachement with cell scrapers. On the other hand, less-adhessive cells might not need EDTA in PBS for detachment.
      2. Thoroughly detach the cells and transfer the cells in PBS into pre-chilled 1.5 ml tubes. Then, rinse the bottom of the well with 500 µl of 3 mM EDTA in PBS and add it to the tube.
      3. Keep cells and tubes always on ice.
    2. Centrifuge the cell suspension at 200 x g at 4 °C for 5 min and then carefully remove the supernatant. Resuspend the pelleted cells in 4-7 volumes of homogenization buffer. For cells from one well of a 6 well plate, we usually add 200 µl of homogenization buffer.
    3. (Optional) Before homogenization, take 10 µl of cell suspension for whole-cell lysate analysis and mix with 30 μl of phosphate-buffered 0.5% Triton X-100/0.5% deoxycholate lysis buffer.
      1. After incubation on ice for 10 min, centrifuge to precipitate the cell debris and carefully aspirate the supernatant as whole-cell lysate.
      2. Add 10 μl of 5x sample buffer and boil for 10 min to prepare "whole-cell lysate" samples without fractionation.
    4. Homogenize the rest of the cells by passing through a 25G ultra-thin-wall needle attached on a 1 ml-syringe until ~90% of cells are disrupted. As extent of destruction of cells is critical for yields of organelles and proteins attached to them, this step has to be carefully evaluated.
      1. At first, checking the status of cells after every 10-15 strokes is recommended. This is done by taking 1-2 µl of homogenate and suspending it into a drop of PBS on the lid of a 6-well plate and observing it on an inverted microscope. Intact naked nuclei which are released from the broken cells are observed as relatively flat and small rounded structures, whereas unbroken cells are obviously larger and more spherical.
      2. How many strokes are necessary for sufficient disintegration of cells can vary between cell types. For example, the cells we are using require more than 30 strokes.
      3. As a note of caution, producing air bubbles should be avoided during the homogenization procedure.   
    5. After cells are sufficiently disintegrated, centrifuge the homogenate at 2,000 x g for 5 min at 4 °C. Carefully take the supernatant as the "post-nuclear fraction" and estimate the volume by marking the level of the homogenate in the pipet tip and measuring the volume of water which fills up to the mark.
      1. Add 1.4 volume of 62% sucrose buffer and mix well until the mixture becomes completely homogeneous. As the two solutions do not easily mix, patienly mix by gentle pipetting with a ‘genomic tip’ (or a pipet tip with tip cut-off) and stirring with the tip at the same time to a point where pipetting does not make "clear streaks" in the solution (de Araujo et al., 2007).
      2. Keep the post-nuclear fraction chilled throughout the procedure.
    6. Place the post-nuclear fraction mixed with 62% sucrose buffer at the bottom of the centrifuge tube and overlay with three layers of sucrose solutions, e.g. 36%, 33% and finally homogenization buffer (8.5%), 1.5 ml each, from bottom to top (Taguchi et al., 2013).
      1. Carefully pour the sucrose buffer not to agitate the interphase too much. Tilt the tube, put the point of the pipette tip just above the surface of the lower layer in the tube and carefully push out the solution in the tip to let it slowly float along the wall.
      2. Intervals between interfaces should be distant enough to avoid contamination from adjacent interfaces during the procedure of harvesting the concentrated organelles.
      3. As optimal sucrose concentrations of the layers for separation of the organelles of interest seem to vary between cell lines, modify the combination of sucrose concentrations according to the distribution of organelle marker proteins until the most suitable combination is found.
    7. Ultra-centrifuge the gradient at 40,000 rpm at 4 °C for 1.5 h. We used a Beckman SW50.1 swing rotor and a Beckman L8-80M ultracentrifuge.
    8. Upon ultra-centrifugation, fractionated organelles are condensed between the layers and visible as “milky-white” bands in interfaces (Figure 1). As the sample presented in the figure was prepared from a 100 mm-dish, the “milky-white” bands are more clearly visible. With N2a cells confluent on a well of 6-well plate, the bands are visible, but less pronounced. Aspirate the "milky bands" as thoroughly as possible. This requires scanning the pipet tip, gradually aspirating, right over a white band in the interface, until it is completely collected. We usually take more volume so that we can most efficiently recover the desired organelles, e.g. 350 µl.


      Figure 1. Example of appearance of fractionated organelles in step-gradient fractionation. The sample was from a 100 mm-cell culture dish and four layers were overlaid on the homogenate. Note that there are four "milky-white" bands at the interfaces of the five layers.

    9. If the purpose is just to evaluate distribution of a specific protein of interest in the fractions, we usually dilute the collected interfaces with 300 μl of PBS with 30 μg of bovine-serum albumin as a carrier protein, and then subject this to methanol/chloroform precipitation (Taguchi et al., 2013). Dissolve the proteins in 1x sample buffer and boil for 10 min to make "fractionated" samples.

Recipes

  1. Phosphate-buffered 0.5% Triton X-100 (TX100)/0.5% deoxycholate (DOC) lysis buffer
    1. First prepare 5% TX100/DOC stock solution
      Triton X-100
      5 ml
      Deoxycholic
      5 g
      Purified water
      up to 100 ml
    2. Then mix 5% TX100/5% DOC, 10x phosphate-buffered saline (PBS) and water in a 50 ml-conical tube
      5% TX100/5% DOC    
      5 ml
      10x PBS        
      5 ml
      Purified water    
      up to 50 ml
  2. 5x sample buffer
    SDS             
    1.2 g
    1 M Tris-HCl (pH7.1)   
    2.5 ml
    Glycerol         
    4 ml
    0.5% BPB         
    300-500 μl
    Water                
    up to 10 ml

Acknowledgments

This protocol was adapted from Taguchi et al. (2013). This work was supported by grants for the National Institute of Health R01 NS076853-01A1 and the Alberta Prion Research Institute (AB, Canada).

References

  1. de Araujo, M. E. and Huber, L. A. (2007). Subcellular fractionation. Methods Mol Biol 357: 73-85.
  2. Taguchi, Y., Mistica, A. M., Kitamoto, T. and Schätzl, H. M. (2013). Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. PLoS Pathog 9(6): e1003466.

简介

在这里,我们介绍了在我们最近的出版物(Taguchi等人,2013)中使用的用于小规模和简单的亚细胞分离的方案,其通过穿过针和蔗糖梯度梯度使用匀浆。
亚细胞分离是一种非常有用的技术,但通常需要大量的细胞。因为我们需要瞬时转染细胞的亚细胞分离,我们开发了用于较小数量细胞的方案。我们的用于亚细胞分级的方案基于deAraújo和Huber(de Araujo等人,2007)公布的方案,尽管根据我们的经验和来自个人通信的信息进行了实质性的修改。由于最佳条件似乎在细胞系之间不同,我们建议进一步修改方案以优化个别实验。我们的方法很简单,但足以分析通过糖基磷脂酰肌醇或其他脂质锚例如朊病毒蛋白锚定到细胞器的内在膜蛋白或蛋白质。然而,非共价连接到膜或细胞器的膜蛋白的蛋白质似乎更容易在制备过程中从细胞器中解离,并且如果这些蛋白质是研究的目的,则可能需要进一步的修饰。
不同于连续梯度,其中感兴趣的蛋白质分散在宽范围内,步梯度分级分离在小规模实验中检测相对少量的蛋白质是有利的,因为它将感兴趣的蛋白质浓缩如果使用蔗糖浓度的适当组合。

关键字:亚细胞分离, 蔗糖梯度, 完整的膜蛋白, 锚定蛋白, 朊病毒蛋白

材料和试剂

  1. Neuro2a细胞(N2a)
  2. 蔗糖(Sigma-Aldrich,目录号:S9378-500G)
  3. 1 M Tris-HCl(pH 7.1)
  4. 0.5M EDTA(Millipore,目录号:324503-1KG)
  5. 纯化水
  6. 100mg/ml环己酰亚胺在DMSO中的溶液(Sigma-Aldrich,目录号:C4859-1ML)
  7. 完全蛋白酶抑制剂混合物(Roche Diagnostics,目录号:04693116001)
  8. 补充有10%胎牛血清的OptiMEM I
  9. 不含钙/镁的磷酸盐缓冲液(Ca/Mg)(Life Technologies,目录号:10010-023)
  10. 脱氧胆酸(Sigma-Aldrich,目录号:D2510-100G)
  11. Triton X-100(Sigma-Aldrich,目录号:93443-100ML)
  12. 十二烷基硫酸钠(Sigma-Aldrich,目录号:L6026-50G)
  13. 甘油(Sigma-Aldrich,目录号:G9012-500ML)
  14. 磷酸盐缓冲的0.5%Triton X-100(TX100)/0.5%脱氧胆酸盐(DOC)裂解缓冲液(参见配方)
  15. 5x样品缓冲液(见配方)

设备

  1. 6孔板
  2. 1ml BD Luer-Lok一次性注射器(BD,目录号:309628)
  3. 25G超薄壁针(Terumo Medical Corporation,目录号:NN-2525R)
  4. 细胞刮刀
  5. 离心管(薄壁,Ultra-Clear TM,5ml,13×51mm)(Beckman Coulter,目录号:344057)
  6. 超速离心机(Beckman Coulter,型号:L8-80M)
  7. 预冷的摇臂转子(Beckman Coulter,型号:SW50.1)
  8. 便携式折射计(可选)(如 ATAGO,型号:PAL-1)
  9. 倒置显微镜

程序

  1. 蔗糖和匀浆缓冲液的制备
    1. 决定阶梯梯度的层的蔗糖浓度,例如36%,33%和8.5%(Taguchi等人,2013)。
    2. 蔗糖缓冲液的浓度基于"重量百分比",即蔗糖浓度(%)= 100×蔗糖(g)/[蔗糖(g)+水 (G)]。
    3. 然后,取需要量的蔗糖并溶解 在纯水中。 通常,测量实际浓度不是 只要蔗糖和水的量是正确的。 如果 必要的,我们用便携式确认实际浓度 折射计。
    4. 在制备蔗糖溶液后, 水,我们测量体积并加入1/250体积的1M Tris-HCl(pH 7.1),使最终浓度为4mM。 我们使用Tris-HCl缓冲液   缓冲器类型应基于以下过程来确定 和实验的目的
    5. 用于均化 缓冲液,匀浆缓冲液的蔗糖浓度为8.5% 并且,除了4mM Tris-HCl,我们添加EDTA至最终 浓度2mM,环己酰亚胺至30μg/ml,以及完全蛋白酶   抑制剂混合物,根据制造商的剂量 指令。 加入环己酰亚胺以保持多核糖体 内质网和增加细胞器的密度 更有效的分离(de Araujo et al。,2007)。

  2. 细胞的均质化
    1. 冲洗N2a细胞,100%汇合在OptiMEM I中补充10% 胎牛血清在6孔板的孔中,一次用预冷冻 PBS,不含Ca/Mg,然后加入700μl的3mM EDTA的PBS溶液,   几分钟,直到细胞可以容易地从底部分离 移液。
      1. 虽然N2a细胞可以用这个程序分离,更多的粘性 细胞可能需要用细胞刮刀机械解开。 上的 另一方面,较少粘附的细胞可能不需要PBS中的EDTA 分离。
      2. 彻底分离细胞并转移细胞   在PBS中倒入预冷的1.5ml管中。 然后,冲洗底部 孔与500μl的3mM EDTA的PBS溶液,并将其加入到试管中。
      3. 保持细胞和管总是在冰上。
    2. 在4℃下将细胞悬浮液以200×g离心5分钟,然后 小心地除去上清液。 在4-7中重悬沉淀的细胞 体积的匀浆缓冲液。 来自6孔的一个孔的细胞 板,我们通常加入200μl匀浆缓冲液。
    3. (可选)均质前,取10μl细胞悬液 全细胞裂解物分析并与30μl磷酸盐缓冲的0.5%   Triton X-100/0.5%脱氧胆酸盐裂解缓冲液。
      1. 在冰上孵育10分钟后,离心沉淀细胞 碎片,并小心地将上清液作为全细胞裂解物吸出。
      2. 加入10μl的5x样品缓冲液,煮沸10分钟,以制备"全细胞裂解液"样品,无需分馏。
    4. 通过通过25G使其余的细胞均匀化 超薄壁针头连接到1ml注射器直到〜90%的细胞 被打乱。 细胞破坏的程度对于产量是至关重要的 的细胞器和蛋白质连接到它们,这一步必须 仔细评估。
      1. 首先,检查单元格的状态 建议每10〜15次。 这是通过取1-2μl 的匀浆并将其悬浮在a的盖子上的一滴PBS中 并在倒置显微镜上观察。 完全裸 观察到从破碎的细胞释放的核 相对平坦和小圆形结构,而未破坏的细胞   明显较大和较为球形。
      2. 多少笔画 细胞充分分裂所必需的细胞可以在细胞之间变化 类型。 例如,我们使用的单元格需要超过30行程。
      3. 作为注意事项,在均质化程序中应避免产生气泡。   
    5. 细胞充分崩解后,离心匀浆 在2,000×g下在4℃温育5分钟。 小心取上清液 "后核分数",并通过标记水平估计体积 在移液管尖端中匀浆并测量水的体积 填补到标记。
      1. 加入1.4体积的62%蔗糖缓冲液, 充分混合直至混合物变得完全均匀。 作为两个 溶液不容易混合,轻轻地用a。轻轻吹打混合 '基因组尖端'(或具有尖端截止的移液管尖端)并与之搅拌 尖端同时到移液不能"清除的点 条纹"(de Araujo et al。,2007)。
      2. 保持后核分数冷冻整个程序。
    6. 将后核部分与62%蔗糖缓冲液混合 底部并用三层蔗糖覆盖 溶液,例如36%,33%,最后是匀浆缓冲液(8.5%),1.5 ml,从底部到顶部(Taguchi等人,2013)。
      1. 小心 倒入蔗糖缓冲液不要搅拌太多的界面。 倾斜   管,将移液管尖端的点放在表面的上方 下层在管中并小心地推出溶液在尖端 让它慢慢地沿着墙壁浮动
      2. 间隔 接口应足够远,以避免相邻的污染   在收获过程中的界面浓缩 细胞器。
      3. 作为层的最佳蔗糖浓度 用于分离感兴趣的细胞器似乎在细胞之间变化 线,修改蔗糖浓度的组合根据   细胞器标记蛋白的分布直到最合适 组合。
    7. 超离心机梯度为40,000 rpm在4℃下1.5小时。 我们使用了Beckman SW50.1摆动转子和a Beckman L8-80M超速离心机。
    8. 超速离心后, 分馏的细胞器在层之间凝聚并且可见 "乳白色"带(图1)。作为样品  该图是从100mm皿制备的,"乳白色"带是  更清晰可见。使N2a细胞在6孔的孔上汇合 板,带是可见的,但不太明显。吸引"乳白色 带"尽可能彻底,这需要扫描移液管尖, 逐渐吸入,在接口的白色带上,直到它  完全收集。我们通常需要更多的音量,以便我们可以 最有效地回收所需的细胞器,例如350μl。


      图 1.分步梯度中分级细胞器的外观的实例 分馏。 样品来自100毫米细胞培养皿和四个 层覆盖在匀浆上。注意有四个 "乳白色"带在五层的界面
    9. 如果目的只是评估特定蛋白质的分布 我们通常稀释收集的界面   与300μl含有30μg牛血清白蛋白作为载体的PBS 蛋白质,然后进行甲醇/氯仿沉淀 (Taguchi et al。,2013)。 将蛋白溶解在1×样品缓冲液中 煮沸10分钟以制备"分馏的"样品。

食谱

  1. 磷酸盐缓冲的0.5%Triton X-100(TX100)/0.5%脱氧胆酸盐(DOC)裂解缓冲液
    1. 首先准备5%TX100/DOC储备溶液
      Triton X-100
      5 ml
      脱氧胆酸
      5克
      纯化水
      最多100 ml
    2. 然后在50ml锥形管中混合5%TX100/5%DOC,10x磷酸盐缓冲盐水(PBS)和水
      5%TX100/5%DOC    
      5 ml
      10x PBS        
      5 ml
      纯净水    
      最多50 ml
  2. 5x样品缓冲液
    SDS             
    1.2克
    1 M Tris-HCl(pH7.1)   
    2.5 ml
    甘油        
    4 ml
    0.5%BPB         
    300-500微升
    水                
    最多10 ml

致谢

该协议改编自Taguchi等人(2013)。 这项工作是由国家卫生研究所R01 NS076853-01A1和艾伯塔朊病毒研究所(AB,加拿大)的拨款支持。

参考文献

  1. de Araujo,M.E。和Huber,L.A。(2007)。 亚细胞分级分离 Methods Mol Biol 357:73- 85.
  2. Taguchi,Y.,Mistica,A.M.,Kitamoto,T.andSchätzl,H.M。(2013)。 螺旋1和2之间的区域对于转化无能朊病毒的有效显性 - 阴性抑制的关键意义 蛋白质。 PLoS Pathog 9(6):e1003466。
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Copyright: © 2014 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. Taguchi, Y. and Schätzl, H. M. (2014). Small-scale Subcellular Fractionation with Sucrose Step Gradient. Bio-protocol 4(11): e1138. DOI: 10.21769/BioProtoc.1138.
  2. Taguchi, Y., Mistica, A. M., Kitamoto, T. and Schätzl, H. M. (2013). Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. PLoS Pathog 9(6): e1003466.
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