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Fractionation by Ultracentrifugation of Gram Negative Cytoplasmic and Membrane Proteins
超速离心法分级分离革兰氏阴性菌细胞质和细胞膜蛋白   

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Abstract

Protein fractionation is a useful separation process which divides membrane proteins (including those located in the outer and inner membrane) and cytoplasmic proteins into discrete fractions. Fractionation of proteins can simplify analysis of the numbers of proteins present, and therefore make easier to characterize any environmentally or mutation induced changes in expression profiles, or changes in protein strucutre resulting from post-translational modification. This protocol is derived from Haigh et al. (2013) and it is specific to Gram negative bacteria.

Materials and Reagents

  1. Bacterial culture media (subject to the needs of the bacterium being studied; for Escherichia coli, Luria Broth or DMEM are suitable)
  2. Tris base (Thermo Fisher Scientific)
  3. Triton X-100 (Sigma-Aldrich) (optional)
  4. Tris-Triton (TT)
  5. HCl (Hydrochloric acid)
  6. TT (see Recipes)

Equipment

  1. Bench top ultracentrifuge (e.g. Beckman Coulter, model: TL-100 ) capable of up to 50,000 rpm for culture volumes of 25 ml or less, and a larger mainframe ultracentrifuge for larger culture volume preparations
  2. Bench top centrifuge capable of up to 12,225 x g
  3. Sonicator
  4. Spectrophotometer

Procedure

  1. The bacteria should be cultured in media appropriate for the bacterial species under investigation or the environmental condition being tested. Cultures can be at logarithmic or stationary phase, although stationary phase cultures can be harder to lyse for protein extraction.
  2. Harvest the bacteria by centrifugation at 6,708 x g for 10 min at 4 °C, discard the culture supernatant (unless it is needed to analyse secreted protein profiles).
  3. Wash the pellet by re-suspending it uniformly with buffer to a volume at 50 times that of the pellet (e.g. the pellet from a 200 ml stationary phase E. coli culture would be around 2 ml in volume and would need at least 100 ml of buffer), then re-centrifuge at 6,708 x g for 10 min at 4 °C. Repeat this wash step.
  4. For separation of E. coli membrane proteins the pellet should be washed in 10 mM Tris base (pH 7.5) (this buffer can be used with any proteins from any cellular source). For small volume culture protein fractionations (e.g. 25 ml) an overnight growth will give an OD600 of approximately 2; once harvested and washed, this would be re-suspended in 0.5-1 ml 10 mM Tris base (pH 7.5), to give a final cell density of OD600 of 20-30 (the suspension should have the appearance of pouring cream). For large volume culture (e.g. 200 ml) re-suspend in 5-10 ml 10 mM Tris base (pH 7.5). If proteolysis is likely to be a problem, a cocktail of broad spectrum protease inhibitors should be included at the levels recommended by the manufacturer.
  5. Freeze the culture suspension at -80 °C for 2 h or overnight to weaken the cell wall and increase the lysis of the bacterial cells by sonication.
  6. Thaw the bacterial suspension on ice and place it in a non-breakable plastic beaker sitting in an ice water bath. Set the sonicator between 6-8 microns and lyse the cells in cycles of 15 sec sonication followed by 45 sec cooling until the lysate changes from an opaque solution into a less turbid solution (usually 4-5 cycles are enough for log phase cells; stationary phase cultures may need more treatments).
  7. Centrifuge the lysed sample at 6,708 x g for 10 min at 4 °C to remove large debris fragments and unlysed cells (these will be contained in the pellet) and transfer the supernatant containing the total protein extract (membrane and cytoplasmic) into appropriate ultracentrifuge tubes.
  8. Ultracentrifuge the protein extract at 108,726 x g for 10 min at 4 °C to separate membrane proteins from cytoplasmic proteins.
  9. After the centrifugation the supernatant will contain the cytoplasmic proteins while the pellet contains the total membrane protein. If only a total membrane proteins fraction is required (comprising both inner and outer membrane proteins) then go to step 11, if only the outer membrane and inner membrane proteins fractions are required go to step 11 and then 10.
  10. The pellet consisting of total membrane proteins is re-suspended in 10 mM Tris-HCl (pH 7.5) supplemented with 2% Triton X-100 in 10 mM Tris-HCl (pH 7.5) (TT; e.g. for a small volume culture initially of 25 ml re-suspend in 100-200 μl of TT); the inner membrane proteins are solubilised by incubation at room temperature with occasional mixing for 30 min. The mixture is then again centrifuged at 108,726 x g for 10 min at 4 °C; the supernatant will contain the inner membrane protein fraction whereas the insoluble pellet will contain the outer membrane protein. To remove any residual inner membrane proteins, the outer membrane pellet is re-extracted for a further 30 min in 500 μl of TT (this step is only to purify the outer membrane proteins and so the supernatant should be discarded).
    Note: The extra wash/extraction steps are needed to increase the relative purity of the different fractions. Sometime it is possible that there are carry over between the fractions especially if people do not take care to remove all supernatants between centrifugations (but that's is more a procedure error). Generally, we determined that two washes are enough to obtain a pure membrane fraction. We tested the purity of the protein fraction by separating them on SDS gel and subjecting the proteins to sequencing. However, more washes steps can be increased to enhance the purity if required.
  11. Wash the total membrane pellet twice or outer membrane pellet once in 10 mM Tris-HCl (pH 7.5); for a small volume culture initially of 25 ml, 500 μl of buffer would be sufficient. Finally, re-suspend in 30-50 μl of the same buffer according to the membrane pellet size.
  12. The concentration of the cytoplasmic and membrane protein samples should be measured using the Bradford assay (He, 2011a) or via a spectrophotometer set at 280 nm.
  13. For storage, proteins should be contained in capped 1.5 or 2 ml sterile plastic tubes, and stored at -20 °C for around 1 month or -80 °C for 6 months or longer. Protein extracts should be thawed on ice and always kept at 0-4 °C for the minimum amount of time to maximize stability and protein activity.
  14. For simple 1-dimensional SDS PAGE, normalized protein samples are mixed with adequate amounts of 2x SDS protein sample buffer and the gel electrophoresis carried out as described by He (2011b) (for 1D SDS-gel electrophoresis). The protein fractions can also be separated by 2D gel electrophoresis if required.

Recipes

  1. TT
    20 μl Triton X-100 in 0.98 ml of 10 mM Tris-HCl (pH 7.5)

Acknowledgments

This protocol is derived from Haigh et al. (2013).

References

  1. He, F. (2011a). Bradford protein assay. Bio-protocol 1(6): e45.
  2. He, F. (2011b). Laemmli-SDS-PAGE. Bio-protocol 1(11): e80.
  3. Haigh, R., Kumar, B., Sandrini, S. and Freestone, P. (2013). Mutation design and strain background influence the phenotype of Escherichia coli luxS mutants. Mol Microbiol 88(5): 951-969.

简介

蛋白分级是一种有用的分离过程,其将膜蛋白(包括位于外膜和内膜中的那些)和胞质蛋白分成离散的级分。 蛋白质的分馏可简化对存在的蛋白质数量的分析,因此使得更容易表征任何环境或突变诱导的表达谱的变化或由翻译后修饰导致的蛋白质结构的变化。 该方案衍生自Haigh等人(2013),并且其特异性针对革兰氏阴性细菌。

材料和试剂

  1. 细菌培养基(根据所研究的细菌的需要;对于大肠杆菌,Luria Broth或DMEM是合适的)
  2. Tris碱(Thermo Fisher Scientific)
  3. Triton X-100(Sigma-Aldrich)(可选)
  4. Tris-Triton(TT)
  5. HCl(盐酸)
  6. TT(见配方)

设备

  1. 台式超速离心机(例如Beckman Coulter,型号:TL-100),对于25ml或更少的培养物体积能够达到50,000rpm,并且较大的主机超速离心机用于更大的培养物体积制备物
  2. 台式离心机,最高可达12,225×g
  3. 超声波仪
  4. 分光光度计

程序

  1. 细菌应在适于所研究的细菌种类或正在测试的环境条件的培养基中培养。 培养物可以是对数或固定相,虽然固定相培养物可能更难溶解用于蛋白质提取。
  2. 通过在4℃下以6,708×g离心10分钟收获细菌,丢弃培养物上清液(除非需要分析分泌的蛋白质谱)。
  3. 通过用缓冲液将其均匀地重悬浮至体积为颗粒的50倍的体积(例如,来自200ml静止期大肠杆菌培养物的沉淀物)来洗涤沉淀物 体积约2ml,并且将需要至少100ml缓冲液),然后在6,708×g下在4℃下再离心10分钟。 重复此洗涤步骤。
  4. 用于分离。大肠杆菌膜蛋白,沉淀应在10mM Tris碱(pH7.5)(该缓冲液可与来自任何细胞来源的任何蛋白质一起使用)中洗涤。对于小体积培养物蛋白质分级分离(例如25ml),过夜生长将给出约2的OD 600;一旦收获并洗涤,将其重悬于0.5-1ml 10mM Tris碱(pH7.5)中,得到OD 600为20-30的最终细胞密度(悬浮液应具有外观的浇注膏)。对于大体积培养(例如200ml),重悬于5-10ml 10mM Tris碱(pH7.5)中。如果蛋白水解可能是一个问题,广谱蛋白酶抑制剂的混合物应包括在制造商推荐的水平。
  5. 在-80℃下将培养物悬浮液冷冻2小时或过夜以削弱细胞壁并通过超声处理增加细菌细胞的裂解。
  6. 将细菌悬浮液在冰上解冻,并将其放置在置于冰水浴中的不可破碎的塑料烧杯中。将超声波仪设置在6-8微米之间,并且在15秒超声处理的循环中裂解细胞,然后冷却45秒,直到裂解物从不透明溶液变成较少混浊的溶液(通常4-5个循环足以用于对数期细胞;静止相培养可能需要更多的治疗)。
  7. 将裂解的样品在6,708×g离心10分钟,以除去大的碎片碎片和未裂解的细胞(这些将包含在沉淀中),并转移含有总蛋白提取物的上清液(膜和细胞质)到合适的超速离心管中。
  8. 在4℃下在108,726×g超速离心蛋白质提取物10分钟,以从细胞质蛋白质中分离膜蛋白质。
  9. 离心后,上清液含有胞质蛋白,而沉淀含有总膜蛋白。如果仅需要总膜蛋白部分(包含内膜蛋白和外膜蛋白),则转到步骤11,如果仅需要外膜和内膜蛋白部分,则进行步骤11,然后进行10。
  10. 将由总膜蛋白组成的沉淀重新悬浮于补充有10mM Tris-HCl(pH 7.5)(TT;例如)的2%Triton X-100的10mM Tris-HCl对于最初为25ml的小体积培养物,在100-200μl的TT中重悬);通过在室温下温育偶尔混合30分钟使内膜蛋白溶解。然后将混合物在4℃下再次在108,726×g离心10分钟;上清液将含有内膜蛋白部分,而不溶性沉淀物将含有外膜蛋白。为了除去任何残留的内膜蛋白,将外膜沉淀在500μlTT中再提取30分钟(该步骤仅用于纯化外膜蛋白,因此上清液应当被丢弃)。
    注意:需要额外的洗涤/提取步骤来提高不同级分的相对纯度。有时,在馏分之间可能存在,特别是如果人们不注意在离心之间去除所有上清液(但是这更多是程序错误)。通常,我们确定两次洗涤足以获得纯膜级分。我们通过在SDS凝胶上分离蛋白质级分并对蛋白质进行测序来测试蛋白质级分的纯度。但是,如果需要,可以增加更多的洗涤步骤以提高纯度。
  11. 在10mM Tris-HCl(pH 7.5)中洗涤总膜沉淀两次或外膜沉淀一次;对于最初为25ml的小体积培养物,500μl缓冲液就足够了。最后,根据膜沉淀大小重悬于30-50μl相同的缓冲液中
  12. 细胞质和膜蛋白样品的浓度应使用Bradford测定法(He,2011a)或通过设定在280nm的分光光度计测量。
  13. 对于储存,蛋白质应该包含在1.5或2毫升无菌塑料管中,并在-20°C下储存大约1个月或-80°C 6个月或更长时间。 蛋白质提取物应在冰上解冻,并始终保持在0-4°C最少的时间,以最大限度地提高稳定性和蛋白质活性。
  14. 对于简单的1维SDS PAGE,将标准化的蛋白质样品与足量的2x SDS蛋白质样品缓冲液混合,并如He(2011b)(用于1D SDS-凝胶电泳)所述进行凝胶电泳。 如果需要,蛋白质级分也可以通过2D凝胶电泳分离

食谱

  1. TT
    20μlTriton X-100的0.98ml 10mM Tris-HCl(pH7.5)中

致谢

该协议来自Haigh等人(2013)。

参考文献

  1. 他,F.(2011a)。 Bradford蛋白测定生物协议 1(6):e45。
  2. 他,F.(2011b)。 Laemmli-SDS-PAGE。生物协议 1(11): e80。
  3. Haigh,R.,Kumar,B.,Sandrini,S。和Freestone,P。(2013)。 突变设计和菌株背景会影响大肠杆菌的表型 luxS 突变体。 Mol Microbiol 88(5):951-969。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sandrini, S. M., Haigh, R. and Freestone, P. P. (2014). Fractionation by Ultracentrifugation of Gram Negative Cytoplasmic and Membrane Proteins. Bio-protocol 4(21): e1287. DOI: 10.21769/BioProtoc.1287.
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ujjwal jit
Panjab University
I am using 50mM HEPES instead of Tris Hcl , using same approach.
4/18/2015 10:02:57 PM Reply