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Small-scale Triton X-114 Extraction of Hydrophobic Proteins
Triton X-114法小规模提取疏水性蛋白   

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参见作者原研究论文

本实验方案简略版
PLOS Pathogens
Jun 2013

Abstract

Here we introduce a protocol for Triton X-114 extraction which we used in our recently-published paper (Taguchi et al., 2013). It is a versatile method to concentrate or partially purify hydrophobic proteins. The presented protocol is based on the protocol published by Bordier (Bordier, 1981) but more simplified and down-scaled for more small-scale and simpler use (Taguchi et al., 2013).

Triton X-114 (TX114) is a non-ionic detergent which has a relatively low clouding point at 22 °C and separates into detergent (Det) and aqueous (Aq) phase at temperatures above the clouding point. During phase separation, hydrophobic solutes in the TX114 solution are sequestered to the Det phase, while hydrophilic solutes are sequestered to the Aq phase. Utilizing this phenomenon, TX114 extraction is a very versatile technique to efficiently concentrate hydrophobic proteins, especially glycosylphosphatidylinositol (GPI)-anchored proteins like the prion protein (PrP), because they have substantial amounts of highly hydrophobic moieties. Besides, phase separation using TX114 tolerates a variety of conditions, e.g. different pH or relatively low concentrations of guanidine hydrochloride. Since the hydrophobic proteins are sequestered to the Det phase as long as the phase separation occurs, and if the hydrophobicity of the protein of interest is not affected by pH or denaturant, this technique can be also utilized to change buffers or to remove denaturants. When using enzymes or proteases which maintain activities in detergent solutions, TX114 can also be used to separate hydrophobic from the water-soluble hydrophilic moieties upon enzymatic digestion of proteins, as done by us using in vitro digestion of PrP with phosphatidylinositol-specific phospholipase C (Taguchi et al., 2013).

Keywords: Triton X-114 extraction (Triton X-114萃取), Hydrophobic protein (疏水性蛋白), GPI-anchored protein (锚定蛋白), Prion protein (朊病毒蛋白), PIPLC digestion (PIPLC消化)

Materials and Reagents

  1. Neuro2a cells (N2a) (100%-confluent on a well of a 6-well plate)
  2. Triton X-114 (Sigma-Aldrich, catalog number: X 114-100 ML )
  3. 10x phosphate-buffered saline (Sigma-Aldrich, catalog number: P5493-1L )
  4. Complete protease-inhibitor cocktail (Roche Diagnostics, catalog number: 04693116001 )
    Note: Optional, depending on the kind of the protein of interest and the purpose of experiments.
  5. 0.5 M EDTA (Millipore, catalog number: 324503-1KG )
  6. Deoxycholic acid (Sigma-Aldrich, catalog number: D2510-100G )
  7. Triton X-100 (Sigma-Aldrich, catalog number: T8787-100 ML )
  8. Sodiumdodecyl sulfate (Sigma-Aldrich, catalog number: L6026-50G )
  9. Glycerol (Sigma-Aldrich, catalog number: G9012-500ML )
  10. Purified water
  11. Phosphate-buffered saline without calcium and magnesium (Ca/Mg) (Life Technologies, catalog number: 10010-023 )
  12. Phosphate-buffered 0.5% Triton X-100 (TX100)/0.5% deoxycholate (DOC) lysis buffer (see Recipes)
  13. 5x sample buffer (see Recipes)

Equipment

  1. Refrigerated centrifuge (max rcf 16,100 x g) (Eppendorf, model: 5415R )
  2. Non-refrigerated centrifuge (max rcf 21,130 x g) (Eppendorf, model: 5424 )
  3. 50 ml conical tube
  4. 6-well plate
  5. 1.5 ml plastic tube

Procedure

  1. Pre-condensation
    This is a step to remove hydrophilic components from the detergent so that the amounts of TX114 which unfavorably sequester to the Aq phase are reduced and, at the same time, the clouding point is lowered. Although dibutyl hydroxytoluene is used in the precondensastion procedure in the original protocol, presumably as an antioxidant to prevent auto-oxidation of TX114, eliminating it from this step does not affect the efficacy of phase-separation. One point to note is that certain concentrations of electrolytes are indispensable for efficient phase separation of TX114. We usually use 1-10 mM Tris-HCl or phosphate buffer, depending on the following experiments. Described here is the specific protocol for our experiments. As this step takes hours, we recommend finishing this step at least by the previous day of harvesting the cells.  
    1. Place 47 ml of buffer in a graduated 50 ml conical tube, then add 3 ml of TX114. As TX114 is very viscous, repeat pipetting until most of the TX114 is homogenized in the buffer. Then, mix well by vortexing or rigorous shaking.
    2. Clarify the solution on ice or 4 °C, then mix again. This usually takes a few minutes.
    3. Incubate the tube at 37 °C to let the solution phase-separate by gravity. As viscous greasy phase which is heavier than water, the Det phase is formed below the Aq phase. Since the concentration of TX114 in the Det phase after phase separation is ~12% (Bordier, 1981), the volume of the detergent phase is expected to be 24 ml. It usually takes a few hours for the top of the Det phase to descend to this level.
    4. When the Det phase falls to the expected level, aspirate the Aq phase and discard it. Then, add fresh buffer up to 50 ml, mix well by vortexing and shaking, and clarify the solution on ice again.
    5. Repeat the steps A3-4, 3-4 times. After the last cycle of phase-separation, discard the Aq phase and store in the dark. The precondensed TX114 can be stored at 4 °C in the dark for at least 6 months.  

  2. Preparation of TX114 lysis buffer and wash buffer
    We usually prepare 2% TX114 lysis buffer. As the concentration of TX114 in the precondensed TX114 (detergent phase) is ~12%, we add one part of the precondensed TX114 to five parts of tris- or phosphate-buffered saline (PBS).
    1. Take 37 ml of purified water in a 50 ml conical tube and add 5 ml of 10x PBS and 8 ml of the precondensed TX114. As viscosity of the solution is lower when the solution is turbid, this step is done at RT. Repeat pipetting until the TX114 sticking to inside the pipette is removed.
    2. Clarify the lysis buffer on ice. If it has already phase-separated, mix well then clear it on ice again.
    3. Take 2.5 ml of 2% TX114 lysis buffer and add to 47.5 ml of buffered saline, e.g. 5 ml of 10x PBS + 42.5 ml of purified water, to make “0.1% TX114 wash buffer”.
    4. Store the lysis buffer and the wash buffer at 4 °C.

  3. Lysis of cells
    As TX114 is a mild detergent, lysis by directly pouring the lysis buffer onto the cells on the plate is inefficient. Therefore, we usually first detach the cells with 3 mM EDTA in Ca/Mg-free PBS, collect the cells in a tube, then lyse them by addition of 2% TX114 lysis buffer.
    1. Remove old culture medium from the N2a cells cultured on a well of a 6-well plate and rinse the monolayer of cells with Ca/Mg-free PBS.
    2. Then, add 3 mM EDTA in Ca/Mg-free PBS (600 µl), and incubate for a few minutes until the cells can be easily detached by pipetting.
      Note: Be careful not to incubate too long. Although our N2a cells can be detached by this procedure, more adhesive cells might require cell scrapers for detachment.
    3. After completely detaching the cells by pipetting, collect the cell suspension in a 1.5 ml plastic tube. Rinse the bottom of the well with 400 µl of 3 mM EDTA in Ca/Mg-free PBS and add it to the tube. Keep the cell suspension on ice all along.
    4. Centrifuge the cell suspension at 1,000 x g, at 4 °C, for 5 min. Then, carefully aspirate and discard the supernatant.
    5. Add 300 µl-1,000 µl of phosphate-buffered 2% TX114 lysis buffer and vortex rigorously for ~30 sec. The volume of the lysis buffer to be added depends on the purpose of experiments and the following procedures.
      1. With larger volumes of lysis buffer, volumes of the Det phase after phase-separations are accordingly larger.
      2. Incubate the cell suspension on ice for ~30 min with vortexing for ~5 sec from time to time.
    6. Centrifuge the cell suspension at 16,100 x g for 2 min at 4 °C to precipitate the cell debris.
      1. Make sure that the temperature of the rotor is low enough not to let the lysate phase-separate during the centrifugation.
      2. Although the clouding point of pure TX114 solution is ~22 °C, it tends to be lower with higher concentrations of impurities.
    7. Carefully take the supernatant to a new tube as the TX114 lysate. 

  4. Phase separation
    1. Incubate the TX114 lysate at 37 °C for ~10 min until the lysate gets turbid and the Det phase partly precipitates.  
    2. Centrifuge at 22,500 x g for 10 min at room temperature. Make sure the rotor is warm enough for efficient phase-separation.
    3. Gently take out the tube and carefully remove the Aq phase. If the hydrophylic proteins in the Aq phase are required for the experiments, put the Aq phase in a new tube.
    4. Add 0.1% TX114 wash buffer and mix well by vortexing and/or shaking. We usually add the same volume as the eliminated Aq phase but different volumes should not cause problems. Then, clarify the lysate on ice.
    5. Incubate the lysate at 37 °C for phase-separation. Repeat steps D1-4 2 more times.

  5. After phase-separation
    We usually precipitate the hydrophobic proteins extracted to the Det phase by methanol/chloroform precipitation (Taguchi et al., 2013) and then add sample buffer and boil for preparation of the samples for SDS-PAGE and immunoblotting.  
    However, other usage of the partially purified hydrophobic proteins is possible, if the detergent can be efficiently removed.

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 (pH 7.1)   
    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. Bordier, C. (1981). Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 256(4): 1604-1607.
  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

在这里我们介绍一个Triton X-114提取的协议,我们在我们最近发表的论文中使用它(Taguchi等人,2013年)。它是集中或部分纯化疏水蛋白的通用方法。所提出的协议基于由Bordier(Bordier,1981)公布的协议,但是对于更小规模和更简单的使用更加简化和缩小(Taguchi等人,2013) > Triton X-114(TX114)是非离子型洗涤剂,在22℃下具有相对低的浊点,在高于浊点的温度下分离成洗涤剂(Det)和水相(Aq)。在相分离期间,TX114溶液中的疏水性溶质被隔离到Det相,而亲水性溶质被隔离到Aq相。利用这种现象,TX114提取是一种非常通用的技术,有效地集中疏水蛋白,特别是糖基磷脂酰肌醇(GPI)锚定蛋白像朊蛋白(PrP),因为他们有大量的高度疏水性部分。此外,使用TX114的相分离可耐受各种条件,例如不同pH或相对低浓度的盐酸胍。由于只要发生相分离,疏水性蛋白就被螯合到Det相,并且如果感兴趣的蛋白质的疏水性不受pH或变性剂的影响,该技术也可以用于改变缓冲液或去除变性剂。当使用在洗涤剂溶液中维持活性的酶或蛋白酶时,TX114也可以用于通过蛋白质的酶消化来分离疏水性和水溶性亲水部分,如通过使用体外PrP消化PrP与磷脂酰肌醇特异性磷脂酶C(Taguchi等人,2013)。

关键字:Triton X-114萃取, 疏水性蛋白, 锚定蛋白, 朊病毒蛋白, PIPLC消化

材料和试剂

  1. Neuro2a细胞(N2a)(在6孔板的孔上100%汇合)
  2. Triton X-114(Sigma-Aldrich,目录号:X114-100ML)
  3. 10x磷酸盐缓冲盐水(Sigma-Aldrich,目录号:P5493-1L)
  4. 完全蛋白酶抑制剂混合物(Roche Diagnostics,目录号:04693116001)
    注意: 可选,取决于感兴趣的蛋白质的种类和实验目的。
  5. 0.5M EDTA(Millipore,目录号:324503-1KG)
  6. 脱氧胆酸(Sigma-Aldrich,目录号:D2510-100G)
  7. Triton X-100(Sigma-Aldrich,目录号:T8787-100ML)
  8. 十二烷基硫酸钠(Sigma-Aldrich,目录号:L6026-50G)
  9. 甘油(Sigma-Aldrich,目录号:G9012-500ML)
  10. 纯化水
  11. 不含钙和镁的磷酸盐缓冲液(Ca/Mg)(Life Technologies,目录号:10010-023)
  12. 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)
  13. 5x样品缓冲液(见配方)

设备

  1. 冷冻离心机(最大rcf 16,100 x g)(Eppendorf,型号:5415R)
  2. 非冷冻离心机(最大rcf 21,130 x g )(Eppendorf,型号:5424)
  3. 50ml锥形管
  4. 6孔板
  5. 1.5 ml塑料管

程序

  1. 预缩合
    这是从洗涤剂中除去亲水性组分的步骤,使得不利地螯合到Aq相的TX114的量减少,同时浊点降低。尽管二丁基羟基甲苯用于原始方案中的预缩合方法中,可能作为抗氧化剂以防止TX114的自氧化,但是从该步骤中消除它不影响相分离的功效。一点要注意的是,某些浓度的电解质对于TX114的有效相分离是不可缺少的。我们通常使用1-10mM Tris-HCl或磷酸盐缓冲液,取决于以下实验。这里描述的是我们的实验的具体方案。由于此步骤需要数小时,我们建议至少在收集细胞的前一天完成此步骤。  
    1. 放置47毫升缓冲液在刻度50毫升锥形管,然后加入3毫升 的TX114。 由于TX114非常粘稠,重复移液,直到大部分 TX114在缓冲液中匀浆。 然后,通过涡旋或充分混合 严重摇晃。
    2. 在冰上或4℃下澄清溶液,然后再次混合。 这通常需要几分钟。
    3. 在37℃下孵育试管,使溶液相分离 重力。 作为比水重的粘性油脂相, 相在Aq相之下形成。 由于TX114的浓度 相分离后的Det相为〜12%(Bordier,1981) 洗涤剂相的体积预期为24ml。 它通常需要一个   几个小时的顶部Det阶段下降到这个水平。
    4. 当Det阶段下降到预期水平时,吸入Aq阶段 并丢弃它。 然后,加入新鲜的缓冲液至50毫升,混匀 涡旋和摇动,并再次在冰上澄清溶液
    5. 重复步骤A3-4,3-4次。 最后一个循环后 相分离,丢弃Aq相并储存在黑暗中。 的 预冷凝的TX114可以在4℃下在黑暗中储存至少6 个月。  

  2. TX114裂解缓冲液和洗涤缓冲液的制备
    我们通常制备2%TX114裂解缓冲液。 由于TX114在预缩合的TX114(洗涤剂相)中的浓度为〜12%,我们将一部分预缩合的TX114加入五份三 - 或磷酸盐缓冲盐水(PBS)中。
    1. 取37毫升纯净水在50毫升锥形管中,加入5毫升10x   PBS和8ml预缩合的TX114。 因为溶液的粘度 当溶液是浑浊时降低,该步骤在室温下进行。 重复 移液,直到TX114粘在移液器内部被移除。
    2. 澄清冰上的裂解缓冲液。 如果它已经相分离,混合好,然后再次在冰上清除。
    3. 取2.5 ml的2%TX114裂解缓冲液,加入47.5 ml缓冲液 盐水,例如5ml 10×PBS + 42.5ml纯净水,使"0.1% TX114洗涤缓冲液"。
    4. 将裂解缓冲液和洗涤缓冲液储存在4℃。

  3. 细胞溶解
    由于TX114是温和的洗涤剂,通过将裂解缓冲液直接倒在板上的细胞上而裂解是无效的。因此,我们通常首先用含Ca/Mg的PBS中的3mM EDTA分离细胞,将细胞收集在管中,然后通过加入2%TX114裂解缓冲液裂解它们。
    1. 从在a的孔上培养的N2a细胞中除去旧培养基 6孔板,用无Ca/Mg的PBS冲洗单层细胞。
    2. 然后,加入3mM EDTA的无Ca/Mg的PBS(600μl),并孵育几个  分钟,直到细胞可以通过移液容易地分离。
      注意: 小心不要孵化太长时间。虽然我们的N2a细胞可以 通过该程序分离,更多的粘附细胞可能需要细胞 刮刀。
    3. 完全分离细胞后 通过吸移,收集细胞悬浮液在1.5ml塑料管中。 用400μl3 mM EDTA在无Ca/Mg的PBS中冲洗孔底部 并将其添加到管中。 保持细胞悬浮在冰上一直。
    4. 在4℃下将细胞悬浮液以1,000×g离心5分钟。 然后,小心地吸出并弃去上清液。
    5. 加入300μl-1,000μl磷酸盐缓冲的2%TX114裂解缓冲液和涡流   严格约30秒。 待加入的裂解缓冲液的体积 取决于实验的目的和以下程序。
      1. 使用较大体积的裂解缓冲液,相分离后Det相的体积因此较大。
      2. 孵育细胞悬浮液在冰上约30分钟,涡旋约5秒的时间。
    6. 在4℃下以16,100×g离心细胞悬浮液2分钟以沉淀细胞碎片。
      1. 确保转子的温度足够低,以使离心过程中裂解物不会相分离。
      2. 虽然纯TX114溶液的浊点为〜22℃,但它趋向于   以较高的杂质浓度降低。
    7. 小心取上清液作为TX114裂解液的新管。

  4. 相分离
    1. 孵育TX114溶胞产物在37°C〜10分钟,直到溶胞产物混浊,Det阶段部分沉淀。  
    2. 在室温下以22,500×g离心10分钟。 确保   转子足够热以有效地进行相分离。
    3. 轻轻地 取出试管,小心地取出Aq相。 如果亲水 蛋白质在Aq期是实验所需的,将Aq 相位在新管中。
    4. 加入0.1%TX114洗涤缓冲液,混匀 通过涡旋和/或摇动。 我们通常添加相同的音量 消除Aq期但不同体积不应该引起问题。 然后,澄清冰上的裂解物
    5. 孵育裂解液在37℃下进行相分离。 重复步骤D1-4 2次。

  5. 相分离后
    我们通常通过甲醇/氯仿沉淀来沉淀提取到Det相的疏水性蛋白质(Taguchi等人,2013),然后加入样品缓冲液并煮沸以制备用于SDS-PAGE和免疫印迹的样品。  
    然而,如果可以有效地除去洗涤剂,则可以使用部分纯化的疏水性蛋白质的其它用途。

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
      纯净水      
      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(pH 7.1)   
    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. Bordier,C。(1981)。 Triton X-114溶液中内在膜蛋白的相分离 J Biol Chem 256(4):1604-1607
  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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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
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 Triton X-114 Extraction of Hydrophobic Proteins. Bio-protocol 4(11): e1139. DOI: 10.21769/BioProtoc.1139.
  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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