搜索

Isolate and Sub-fractionate Cell Membranes from Caulobacter crescentus
新月柄杆菌细胞膜的分离和细分级   

下载 PDF 引用 收藏 1 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Molecular Microbiology
Jun 2012

Abstract

Cell membranes from Caulobacter can be isolated and separated into inner and outer membranes according their characteristic buoyant densities on a sucrose gradient. Fractionation can be used to determine the localisation of uncharacterised proteins and to enrich protein complexes present in either of these membranes for biochemical analysis such blue-native PAGE and immunoprecipitation.

Keywords: Bacteria (细菌), Membrane (膜), Density fractionation (密度分离), Caulobacter (caulobacter)

Materials and Reagents

  1. 0.2% Bacto Peptone
  2. 0.1% yeast extract
  3. MgSO4
  4. CaCl2
  5. Tris (Sigma-Aldrich)
  6. Sucrose (Sigma-Aldrich)
  7. Lysozyme (Sigma-Aldrich)
  8. EDTA-free protease inhibitors (F. Hoffmann-La Roche, catalog number: 11873580001 )
  9. Dnase I (Sigma-Aldrich, catalog number: DN25 )
  10. MgCl2 (Sigma-Aldrich)
  11. EDTA (Sigma-Aldrich)
  12. Na2HPO4
  13. KH2PO4
  14. NH4Cl
  15. FeSO4 (EDTA chelate) (Sigma-Aldrich, catalog number: F0518 )
  16. Glucose
  17. Spheroplasting buffer
  18. Growth media (Rich PYE or Minimal M2G) (see Recipes)
  19. Spheroplasting buffer (see Recipes) 
  20. Lysis buffer (see Recipes)
  21. Growth media (Rich PYE or Minimal M2G) (see Recipes)
  22. Sucrose solutions (see Recipes)
  23. TEM buffer (see Recipes)

Equipment

  1. Centrifuges
  2. Peristaltic pump (Bio-Rad Laboratories, catalog number: 731-8140EDU )
  3. High-pressure homogenizer (Avestin Emulsiflex, EmulsiFlex-C5)
  4. 7 and 15 ml Dounce homogenizers (WHEATON)
  5. BECKMAN centrifuge, fixed angle rotor (Ti45 or Ti60), swing-out rotor (SW40) and appropriate tubes
  6. Density gradient fractionation system (Brandel, model: BR188 )
  7. Beckman Ultra-Clear tubes
  8. SW40 rotor tubes

Procedure

  1. Preparation of membranes
    1. Grow Caulobacter in PYE media at 30 °C till OD660 is 0.6-0.8.
    2. Spin 2 L of Caulobacter culture at 10,000 x g for 12 min at 4 °C. For all following steps, keep everything on ice.
    3. Resuspend cell pellet with spheroplasting buffer. The volume of spheroplasting buffer required (in ml) = [(OD660 x ml culture volume)/100] x 7. For example, a cell pellet from a 2 L culture with an OD660 of 0.8 will be resuspended by 112 ml of spheroblasting buffer.
    4. Add 100 mg/ml lysozyme (dissolved in spheroplasting buffer). Leave on ice for 2 min. For cultures grown in M2G minimal media, use less lysozyme (50 mg/ml) as cell grown under these conditions have weaker cell walls. M2G minimal media can be used to examine differential expression of outer membrane proteins as a result of carbon starvation.
    5. Add 1 tablet of protease inhibitor cocktail, 25 μg/ml Dnase I (final concentration) and 10 mM MgCl2 (final concentration).
    6. Add 2 volumes of cold lysis buffer to the cells. This must be done slowly to avoid localised cell lysis. Add the lysis buffer at a rate of 4-6 ml/min, either drop-wise with a pipette or using a peristaltic pump. The cell solution must be constantly swirled during the addition of lysis buffer to avoid localised lysis.
    7. Completely lyse the cells with the Avestin Emulsiflex using homogenizing pressure set to ~12,000-15,000 psi.
    8. Pellet insoluble debris at 10,000 x g, 20 min at 4 °C. Collect the supernatant (contains membranes).
    9. Spin the supernatant in the Beckman ultracentrifuge using the Ti45 rotor (36,000 rpm, 1.5 h, 4 °C). After the spin, keep the membrane pellet and discard the supernatant.
    10. To wash membranes, resuspend the membrane pellets in 50-100 ml of spheroblasting buffer and repeat step 9. Addition of 1x protease inhibitor cocktail to the spheroblasting buffer for the wash step is optional.
    11. Resuspend the membrane pellet using ~ 6-7 ml of 25% sucrose (w/v). Addition of 1x protease inhibitor cocktail to the sucrose solution is optional but unnecessary.

  2. Fractionation of cell membranes
    1. Prepare the sucrose gradients using the Beckman Ultra-Clear tubes. Use Beckman tubes (14 x 95 mm) for the SW40 rotor. The sucrose gradient consists of 1.7 mL each of 60, 55, 50, 45, 40, 35, 30% sucrose. To pour the sucrose gradient, pipette the required volume for each sucrose concentration into an eppendorf tube. Using a plastic pasteur pipette, transfer 60% sucrose solution from the eppendorf tube to the bottom of the Ultra-clear tube. Hold the Ultra-clear tube at 45° angle before transferring the 55% sucrose solution by slowly adding it drop-by-drop near the 60% sucrose solution. Then add 50, 45, 40, 35 and 30% sucrose. I found this particular method minimised disruption to the gradient.
    2. Add ~ 1.2 ml of membrane sample (from step 10) on top of the sucrose gradient. Make sure to balance the sucrose gradient tubes with the SW40 rotor tubes.
    3. Spin sucrose gradients for 40 h in a SW40 rotor at 34,000 rpm, 4 °C to fractionate the membranes by equilibrium sedimentation. Use slow break so the sucrose gradient is maintained.
    4. The gradient should have a yellow-brown region (inner membranes) and a cloudy white region (outer membranes). Isolate 0.5 ml fractions using the Brandel/Foxy Jr fractionator and 2 M sucrose (in 5 mM EDTA, pH 7.5) as the displacing fluid. Alternatively, collect 0.5 ml fractions from the top of the gradient. Fractions 9-12 generally contain inner membranes and fractions 16-18 contain outer membranes from a total of approximately 23 fractions. To confirm, perform western blot analysis using an antibody against an inner and an outer membrane protein (see figure below showing 20 μl of each fraction for SDS-PAGE and analysis by Coomassie; and 5 μl of each fraction analysed by western blotting using our in-house antibodies).
    5. Freeze fractions in liquid N2 and store at -80 °C. For further analysis, pool relevant fractions and wash in TEM buffer before resuspending membranes in the buffer of choice. To wash, mix fractions with at least 5 volumes of TEM buffer and spin in the Beckman ultracentrifuge (36,000 rpm, 1.5 h, 4°C).


      Figure 1. Membranes from wild-type C. crescentus were fractionated on sucrose gradient and analysed by SDS-PAGE. Coomassie Brilliant Blue staining (upper panel) reveals separation of the membrane protein profiles and immunoblotting (lower panel) for the inner membrane protein TimA and the outer membrane protein BamA.

Recipes

  1. Growth media (Rich PYE or minimal M2G)
    PYE (peptone yeast extract) broth
    0.2% Bacto peptone
    0.1% yeast extract
    1 mM MgSO4
    0.5 mM CaCl2
    M2G (M2 minimal salt medium with glucose as the sole carbon source)
    6.1 mM Na2HPO4
    3.9 mM KH2PO4
    9.3 mM NH4Cl
    0.5 mM MgSO4
    10 uM FeSO4 (EDTA chelate)
    0.5 mM CaCl2
    0.2% glucose
  2. Spheroplasting buffer
    10 mM Tris (pH 7.5)
    0.75 M sucrose
  3. Lysis buffer
    1.5 mM EDTA (pH 7.5)
  4. Sucrose solutions
    25% sucrose (w/v) in 5 mM EDTA (pH 7.5)
    60, 55, 50, 45, 40, 35, 30% (w/v) sucrose in 5 mM EDTA (pH 7.5)
    2 M sucrose displacing fluid for fractionation
  5. TEM buffer
    10 mM Tris (pH 7.5)
    1 mM EDTA
    10 mM MgCl2
    10% glycerol

Acknowledgments

The author acknowledges the help and support of Professor Trevor Lithgow and Dr. Xenia Gatsos.

References

  1. Anwari, K., Poggio, S., Perry, A., Gatsos, X., Ramarathinam, S. H., Williamson, N. A., Noinaj, N., Buchanan, S., Gabriel, K., Purcell, A. W., Jacobs-Wagner, C. and Lithgow, T. (2010). A modular BAM complex in the outer membrane of the alpha-proteobacterium Caulobacter crescentus. PLoS One 5(1): e8619.
  2. Anwari, K., Webb, C. T., Poggio, S., Perry, A. J., Belousoff, M., Celik, N., Ramm, G., Lovering, A., Sockett, R. E., Smit, J., Jacobs-Wagner, C. and Lithgow, T. (2012). The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex. Mol Microbiol 84(5): 832-844.

简介

来自弯曲杆菌的细胞膜可以根据蔗糖梯度上的特征浮力密度分离并分离成内膜和外膜。 分馏可以用于确定未表征的蛋白质的定位并富集存在于这些膜的任一个中的蛋白质复合物用于生物化学分析,例如蓝色天然PAGE和免疫沉淀。

关键字:细菌, 膜, 密度分离, caulobacter

材料和试剂

  1. 0.2%细菌蛋白胨
  2. 0.1%酵母提取物
  3. MgSO 4 4 /
  4. CaCl <2>
  5. Tris(Sigma-Aldrich)
  6. 蔗糖(Sigma-Aldrich)
  7. 溶菌酶(Sigma-Aldrich)
  8. 无EDTA蛋白酶抑制剂(F.Hoffmann-La Roche,目录号:11873580001)
  9. Dnase I(Sigma-Aldrich,目录号:DN25)
  10. MgCl 2(Sigma-Aldrich)
  11. EDTA(Sigma-Aldrich)
  12. Na HPO 4
  13. KH 2 PO 4
  14. NH 4 Cl
  15. FeSO 4(EDTA螯合物)(Sigma-Aldrich,目录号:F0518)
  16. 葡萄糖
  17. 球形缓冲液
  18. 生长培养基(Rich PYE或Minimal M2G)(参见配方)
  19. 球形缓冲液(见配方)
  20. 裂解缓冲液(见配方)
  21. 生长培养基(Rich PYE或Minimal M2G)(参见配方)
  22. 蔗糖溶液(见配方)
  23. TEM缓冲液(参见配方)

设备

  1. 离心机
  2. 蠕动泵(Bio-Rad Laboratories,目录号:731-8140EDU)
  3. 高压均化器(Avestin Emulsiflex,EmulsiFlex-C5)
  4. 7和15ml Dounce匀浆器(WHEATON)
  5. BECKMAN离心机,定角转子(Ti45或Ti60),旋出转子(SW40)和适当的管子
  6. 密度梯度分馏系统(Brandel,型号:BR188)
  7. Beckman超清洁管
  8. SW40转子管

程序

  1. 膜的制备
    1. 在30℃下在PYE培养基中生长弯曲杆菌,直到OD <660> 为0.6-0.8。
    2. 在4℃下以10,000×g离心12分钟,使2L的杆菌属培养物转染。对于所有后续步骤,请保持一切冰。
    3. 用球形沉淀缓冲液重悬细胞沉淀。所需的球形化造粒缓冲液的体积(以ml计)= [(OD 660xml培养物体积)/100]×7。例如, 660的0.8将由112ml球化缓冲液重悬浮
    4. 加入100mg/ml溶菌酶(溶解在球形造粒缓冲液中)。在冰上放置2分钟。对于在M2G基本培养基中生长的培养物,使用较少的溶菌酶(50mg/ml),因为在这些条件下生长的细胞具有较弱的细胞壁。 M2G最小培养基可用于检查由于碳饥饿导致的外膜蛋白的差异表达
    5. 加入1片蛋白酶抑制剂混合物,25μg/ml Dnase I(终浓度)和10mM MgCl 2(终浓度)。
    6. 加入2体积的冷裂解缓冲液的细胞。这必须缓慢进行,以避免局部细胞裂解。以4-6 ml/min的速率加入裂解缓冲液,用移液管或使用蠕动泵滴加。在加入裂解缓冲液期间,细胞溶液必须不断涡旋以避免局部裂解
    7. 使用均匀化压力设置为〜12,000-15,000psi用Avestin Emulsiflex完全裂解细胞。
    8. 颗粒不溶碎片在10,000×g下,在4℃下20分钟。 收集上清液(含膜)。
    9. 使用Ti45转子(36,000rpm,1.5h,4℃)在Beckman超速离心机中旋转上清液。 旋转后,保留膜沉淀并弃去上清液
    10. 为了洗涤膜,将膜沉淀重悬在50-100ml球化缓冲液中并重复步骤9.向洗涤步骤中的球化缓冲液中加入1x蛋白酶抑制剂混合物是可选的。
    11. 使用〜6-7ml的25%蔗糖(w/v)重悬浮膜沉淀。 向蔗糖溶液中加入1x蛋白酶抑制剂混合物是可选的,但不是必需的

  2. 细胞膜分级
    1. 使用Beckman Ultra-Clear管制备蔗糖梯度。使用Beckman管(14 x 95 mm)作为SW40转子。蔗糖梯度由各自为1.7mL的60,55,50,45,40,35,30%蔗糖组成。为了倾倒蔗糖梯度,将每种蔗糖浓度所需的体积移至埃彭道夫管中。使用塑料巴斯德吸管,将60%蔗糖溶液从eppendorf管转移到超透明管的底部。将超透明管保持在45°角,然后通过在60%蔗糖溶液附近缓慢地逐滴加入来转移55%蔗糖溶液。然后加入50,45,40,35和30%蔗糖。我发现这个特定的方法最小化了对渐变的破坏
    2. 在蔗糖梯度上加入约1.2ml膜样品(来自步骤10)。确保蔗糖梯度管与SW40转子管平衡。
    3. 在SW40转子中以34,000rpm,4℃旋转蔗糖梯度40小时,以通过平衡沉降分离膜。使用缓慢断裂,以保持蔗糖梯度。
    4. 梯度应该具有黄棕色区域(内膜)和多云白色区域(外膜)。使用Brandel/Foxy Jr分级器和2M蔗糖(在5mM EDTA,pH7.5)作为置换液分离0.5ml级分。或者,从梯度的顶部收集0.5ml级分。级分9-12通常含有内膜,级分16-18含有总计约23个级分的外膜。为了证实,使用针对内膜蛋白和外膜蛋白的抗体进行蛋白质印迹分析(参见下图,显示20μl的每个级分用于SDS-PAGE和通过考马斯分析;和5μl的每个级分通过使用我们的家兔抗体)。
    5. 冷冻液体N 2中的馏分并储存在-80℃。为了进一步分析,将相关级分汇集并在TEM缓冲液中洗涤,然后将膜重悬浮在所选择的缓冲液中。洗涤,用至少5体积的TEM缓冲液混合级分,并在Beckman超速离心机(36,000rpm,1.5小时,4℃)中旋转。

      图1.野生型 C的膜。 crescentus 在蔗糖梯度上分级并通过SDS-PAGE分析。 考马斯亮蓝染色(上图)揭示了内膜蛋白TimA和外膜蛋白BamA的膜蛋白质图谱和免疫印迹分离(下图)。

食谱

  1. 生长培养基(富PYE或最小M2G)
    PYE(胨酵母提取物)肉汤
    0.2%细菌蛋白胨 0.1%酵母提取物
    1mM MgSO 4 0.5mM CaCl 2·h/v M2G(以葡萄糖为唯一碳源的M2最小盐培养基)
    6.1mM Na 2 HPO 4
    3.9mM KH 2 PO 4 sub/
    9.3mM NH 4 Cl/s 0.5mM MgSO 4 10μMFeSO 4(EDTA螯合物)
    0.5mM CaCl 2·h/v 0.2%葡萄糖
  2. 球形缓冲液
    10mM Tris(pH7.5) 0.75 M蔗糖
  3. 裂解缓冲液
    1.5mM EDTA(pH7.5)
  4. 蔗糖溶液
    25%蔗糖(w/v)的5mM EDTA(pH7.5)中 60,55,50,45,40,35,30%(w/v)蔗糖的5mM EDTA(pH7.5)中的溶液。
    2 M蔗糖置换液进行分馏
  5. TEM缓冲区
    10mM Tris(pH7.5) 1mM EDTA
    10mM MgCl 2/
    10%甘油

致谢

作者承认Trevor Lithgow教授和Xenia Gatsos博士的帮助和支持。

参考文献

  1. Anwari,K.,Poggio,S.,Perry,A.,Gatsos,X.,Ramarathinam,SH,Williamson,NA,Noinaj,N.,Buchanan,S.,Gabriel,K.,Purcell,AW,Jacobs-Wagner ,C。和Lithgow,T。(2010)。 在α-变形杆菌属新月柄杆菌外膜中的模块化BAM复合物。 PLoS One 5(1):e8619。
  2. Anwari,K.,Webb,CT,Poggio,S.,Perry,AJ,Belousoff,M.,Celik,N.,Ramm,G.,Lovering,A.,Sockett,RE,Smit,J.,Jacobs-Wagner ,C。和Lithgow,T。(2012)。 细菌外膜BAM复合物的新脂蛋白亚基的进化。 Mol Microbiol 84(5):832-844。
  • English
  • 中文翻译
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: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Anwari, K. (2012). Isolate and Sub-fractionate Cell Membranes from Caulobacter crescentus. Bio-protocol 2(20): e276. DOI: 10.21769/BioProtoc.276.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。

June Javens-Wolfe
Indiana University
Hello,

I've been trying to use this protocol to sub-fractionate membranes from Caulobacter crescentus, but have not had good luck so far. I'm not getting very clean separation. The inner membrane fraction is found over a wide range of fractions (almost the whole gradient). The outer membranes are confined to a smaller number of fractions, though. It seems like both fractions are ending up pretty close to the bottom of the tube, in denser fractions than what you normally get.

I had initially been using the CB15 strain, but I have been getting the same results with the CB15N strain.

I was wondering if you had ever come across this problem, and if you knew of a way to correct it. Do you think that the lysis conditions could be to blame? I've been using a Microfluidics microfluidizer to lyse the spheroplasts. Any tips you can give me would be greatly appreciated.

Thank you!

June
8/19/2013 1:55:13 PM Reply
Khatira Anwari
Monash University

Hi June,

After reading about your problem, the first thought was that the sucrose gradient has been disturbed. This can happen mainly when it is being prepared as you stack sucrose solutions to make the gradient or when the sucrose gradients are centrifuged. After you pour the gradients, you should be able to delineate the different concentrations of sucrose by holding the tube to light to see the translucent boundaries. Also, before you spin the sucrose gradients at 34,000 rpm in the ultracentrifuge, make sure you set the acceleration/deceleration to a slow speed. On the Beckman ultracentrifuge that I was using, I would set the acceleration/deceleration speed to 5 (with 9 being the maximum). This ensures that the sucrose gradients are not disrupted by sudden high speeds. Considering your inner membranes and outer membranes are near the bottom of the tube, it is likely the run is started or stopped too quickly.

I do not think the lysis conditions are too blame, as long as you are using somewhat consistent pressure to bust the spheroblasts.

Cheers,
Khatira


8/19/2013 7:41:22 PM


June Javens-Wolfe
Indiana University

Thanks! I'll try that. I had set the deceleration pretty low, but I didn't adjust the acceleration speed.

8/20/2013 7:32:25 AM