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Immunolabeling of Proteins in situ in Escherichia coli K12 Strains
在大肠杆菌K12菌株中进行蛋白原位免疫标记   

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Abstract

This protocol was developed to label proteins in bacterial cells with antibodies conjugated to a fluorophore for fluorescence microscopy imaging. The procedure is optimized to minimize morphological changes and also to minimize the amount of antibodies needed for the staining. The protocol can also be used with primary antibodies conjugated to a fluorophore. The method has been verified extensively (van der Ploeg et al., 2013), but it should be noted that one case in Caulobacter crescentus (Hocking et al., 2012) has been reported in which the localization of a protein changed upon fixation by formaldehyde/glutaraldehyde. However, the localization of the same protein in E. coli did not change.

Keywords: Protein localization(蛋白定位), Fixation(固定), Imaging(成像), Fluorescence(荧光), Bacteria(菌)

Materials and Reagents

  1. Gram-negative bacteria (the protocol is developed for Escherichia coli, but it also works on other species)
  2. Formaldehyde (FA) (Sigma-Aldrich, catalog number: 47608 )
  3. Glutaraldehyde (GA) (Merck KGaA, catalog number: 1-04239-0250 )
  4. Tween-20 (Sigma-Aldrich, catalog number: P9416-100ml )
  5. Triton X-100 (Merck KGaA, catalog number: 1.08643.1000 )
  6. EDTA (Sigma-Aldrich, catalog number: ED255 )
  7. Lysozyme (Sigma-Aldrich, catalog number: L6876 )
    Note: The lysozyme is dissolved at 100 μg/ml in the PBS pH 7.2 with 5 mM EDTA ready to use 1 ml aliquots and stored at -20 °C. After using it the leftover is discarded.
  8. Blocking reagents (F. Hoffmann-La Roche, catalog number: 1096176 ))
  9. Cy3-AffiniPure Donkey Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch, catalog number: 711-165-152 )
    Note: Minimal cross-reaction to Bovine, Chicken, Goat, Guinea, Pig, Syrian Hamster, Horse, Human, Mouse, Rat and Sheep serum proteins. The in buffer freeze-dried Cy3 labeled secondary antibodies are dissolved in H2O to a final concentration of 1.5 mg/ml and aliquoted as 20 μl samples. Once thawed the secondary antibodies are stored at 4 °C. After one month take a new sample from the -20 °C.
  10. PBS buffer (pH 7.2) (see Recipes)

Equipment

  1. Shaking incubator to grow bacteria
  2. 500 μl or 1 ml tubes (Eppendorf)
  3. 50 ml Tubes (Greiner Bio-One GmbH, catalog number: 227261 ) (Alternative Sorval SS34 tubes)
  4. Eppendorf centrifuge 5804 R (Alternative Sorval centrifuge for SS32 rotor)
  5. Eppendorf centrifuge (cooled)
  6. Shaking incubation block for eppendorf tubes

Procedure

  1. Permeabilization of the cells
    1. Escherichia coli cells (LMC500 strains) are grown in medium at 28-42 °C and fixed in 2.8% FA and 0.04% GA as follows: 12.2 ml culture with OD450 of 0.2 (or OD600 of 0.3) is mixed by addition of a pre-mixture of 1 ml 37% FA and 21 μl 25% GA while shaking in the water bath used for growth. Transfer the culture to 50 ml Greiner centrifuge tubes.
      Note: It is recommended keeping the OD600 below 0.3 for optimal exponential growth in rich medium and the OD450 below 0.2 for minimal medium.
    2. Incubate15 min at room temperature (RT) standing and centrifuge at 4,000 x g for 10 min at RT.
    3. Wash the cells once in 1 volume PBS (pH 7.2).
    4. Resuspend the pellet in 150 μl PBS pH 7.2 and transfer the cells to 500 μl Eppendorf tubes.
    5. Pellet the cells by centrifugation at 4,500 x g (7,000 rpm) for 5 min (RT or 4 °C) and wash twice in 150 μl PBS (pH 7.2). The cells can be stored up to a month at 4 °C.
    6. All subsequent steps are performed in 150 μl (less is also possible) and all centrifugation steps are at 4,500 x g (7,000 rpm) for 5 min at RT or 4 °C.
    7. Incubate the cells in 0.1% Triton X-100/PBS pH 7.2 standing for 45 min at RT.
    8. Wash the cells three times in PBS (pH 7.2).
    9. Incubate the cells in PBS (pH 7.2) containing 100 μg/ml lysozyme and 5 mM EDTA for 45 min (or 30 min in case of cell wall mutants) at room temperature.
    10. Wash the cells three times in PBS (pH 7.2).

  2. Labeling procedure
    1. Block non-specific binding sites by incubating the cells standing or shaking in 0.5% (w/v) blocking reagents in PBS (pH 7.2) for 30 min at 37 °C.
    2. Incubate with primary antibody (rule of thumb 10 times more concentrated than needed for immunoblotting) diluted in blocking buffer, 1-2 h at 37 °C in shaking incubator (minimal incubation time 30 min, maximal incubation time over night at 4 °C depending on the antibodies).
    3. Wash the cells three times in PBS (pH 7.2) containing 0.05% (v/v) Tween-20.
    4. Incubate with secondary antibody donkey-α-rabbit-CY3 (guarantee no cross reactivity against E. coli) diluted in blocking buffer (1:600) for 30 min at 37 °C.
      Note: Centrifuge the antibody in blocking solution for 1 min at max speed to remove clumps of dye before adding it to the cells.
    5. Wash the cells three times in 150 μl PBS (pH 7.2)/0.05% Tween-20.
    6. Wash the cells once in in 150 μl PBS.
    7. Resuspend the cells in PBS.
      Notes:
      1. Adjust the volume to the amount of cells (usually 20 μl), i.e. the cell concentration should be high enough for the microscopic analysis.
      2. Antisera against E. coli proteins can very conveniently be separated from contaminating IgG by incubating the serum against a strain that has the gene of interest deleted using the same procedure as above. Subsequently the non-bound IgG is used for the incubation with the wild type strain. If the protein of interest is essential, the serum has to be affinity purified against the pure protein of interest.

Notes

Fixation of the bacterial culture (either by formaldehyde/glutaraldehyde or by ethanol or methanol), which is essential for the immunolabelling procedure, gives an osmotic shock to the cells. The localization of membrane bound or membrane associated proteins or of cytosolic proteins is not affected by the osmotic shock. However, freely in the periplasm diffusing proteins can be shocked to the cell poles during fixation. Therefore, we do advise to verify the localization of periplasmic freely diffusing protein by analysis of the localization of fluorescent protein fusions to these proteins in combination with life imaging.

Recipes

  1. PBS buffer (pH 7.2) (per L)
    140 mM NaCl
    27 mM KCl
    10 mM Na2HPO4.2H2O
    2 mM KH2PO4
    Note: PBS should always be super-sterile.

Acknowledgments

The protocol described has been used in the following publications: Blaauwen et al. (1999); Aarsman et al. (2005); Potluri et al. ( 2010); Typas et al. (2010); Banzhaf et al. (2012); van der Ploeg et al. (2013) and Egan et al. (2014).

References

  1. Aarsman, M. E., Piette, A., Fraipont, C., Vinkenvleugel, T. M., Nguyen-Disteche, M. and den Blaauwen, T. (2005). Maturation of the Escherichia coli divisome occurs in two steps. Mol Microbiol 55(6): 1631-1645.
  2. Banzhaf, M., van den Berg van Saparoea, B., Terrak, M., Fraipont, C., Egan, A., Philippe, J., Zapun, A., Breukink, E., Nguyen-Disteche, M., den Blaauwen, T. and Vollmer, W. (2012). Cooperativity of peptidoglycan synthases active in bacterial cell elongation. Mol Microbiol 85(1): 179-194.
  3. Den Blaauwen, T., Buddelmeijer, N., Aarsman, M. E., Hameete, C. M. and Nanninga, N. (1999). Timing of FtsZ assembly in Escherichia coli. J Bacteriol 181(17): 5167-5175.
  4. Egan, A. J., Jean, N. L., Koumoutsi, A., Bougault, C. M., Biboy, J., Sassine, J., Solovyova, A. S., Breukink, E., Typas, A., Vollmer, W. and Simorre, J. P. (2014). Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B. Proc Natl Acad Sci U S A 111(22): 8197-8202.
  5. Hocking, J., Priyadarshini, R., Takacs, C. N., Costa, T., Dye, N. A., Shapiro, L., Vollmer, W. and Jacobs-Wagner, C. (2012). Osmolality-dependent relocation of penicillin-binding protein PBP2 to the division site in Caulobacter crescentus. J Bacteriol 194(12): 3116-3127.
  6. Potluri, L., Karczmarek, A., Verheul, J., Piette, A., Wilkin, J. M., Werth, N., Banzhaf, M., Vollmer, W., Young, K. D., Nguyen-Disteche, M. and den Blaauwen, T. (2010). Septal and lateral wall localization of PBP5, the major D,D-carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment. Mol Microbiol 77(2): 300-323.
  7. Typas, A., Banzhaf, M., van den Berg van Saparoea, B., Verheul, J., Biboy, J., Nichols, R. J., Zietek, M., Beilharz, K., Kannenberg, K., von Rechenberg, M., Breukink, E., den Blaauwen, T., Gross, C. A. and Vollmer, W. (2010). Regulation of peptidoglycan synthesis by outer-membrane proteins. Cell 143(7): 1097-1109.
  8. van der Ploeg, R., Verheul, J., Vischer, N. O., Alexeeva, S., Hoogendoorn, E., Postma, M., Banzhaf, M., Vollmer, W. and den Blaauwen, T. (2013). Colocalization and interaction between elongasome and divisome during a preparative cell division phase in Escherichia coli. Mol Microbiol 87(5): 1074-1087. 

简介

该协议被开发来标记细菌细胞中的蛋白质与缀合到荧光显微镜成像荧光的抗体。 优化程序以使形态变化最小化,并且使染色所需的抗体的量最小化。 该方案还可以与结合至荧光团的一抗一起使用。 该方法已经广泛地验证(van der Ploeg等人,2013),但是应当注意的是,在新月柄杆菌中的一种情况(Hocking等人,/2012>),其中蛋白质的定位在通过甲醛/戊二醛固定时改变。 然而,相同蛋白质在E中的定位。 大肠杆菌没有改变。

关键字:蛋白定位, 固定, 成像, 荧光, 菌

材料和试剂

  1. 革兰氏阴性菌(该方案是为大肠杆菌开发的,但它也适用于其他物种)
  2. 甲醛(FA)(Sigma-Aldrich,目录号:47608)
  3. 戊二醛(GA)(Merck KGaA,目录号:1-04239-0250)
  4. 吐温-20(Sigma-Aldrich,目录号:P9416-100ml)
  5. Triton X-100(Merck KGaA,目录号:1.08643.1000)
  6. EDTA(Sigma-Aldrich,目录号:ED255)
  7. 溶菌酶(Sigma-Aldrich,目录号:L6876) 注意:将溶菌酶以100μg/ml溶解在含有5mM EDTA的PBS pH7.2中,准备使用1ml等分试样并储存在-20℃下。
  8. 封闭试剂(F.Hoffmann-La Roche,目录号:1096176))
  9. Cy3-AffiniPure驴抗兔IgG(H + L)(Jackson ImmunoResearch,目录号:711-165-152)
    注意:对牛,鸡,山羊,几内亚,猪,叙利亚仓鼠,马,人,小鼠,大鼠和绵羊血清蛋白的最小交叉反应。将缓冲液冷冻干燥的Cy3标记的二抗溶解于H 2 O中至终浓度为1.5mg/ml,并等分为20μl样品。一旦解冻,二抗在4℃下储存。一个月后从-20°C取新的样品。
  10. PBS缓冲液(pH 7.2)(参见配方)

设备

  1. 振荡孵化器以生长细菌
  2. 500μl或1ml管(Eppendorf)
  3. 50ml管(Greiner Bio-One GmbH,目录号:227261)(备选Sorval SS34管)
  4. Eppendorf离心机5804 R(SS32转子的备用离心机)
  5. Eppendorf离心机(冷却)
  6. 摇匀培养箱用于eppendorf管

程序

  1. 细胞的渗透
    1. 大肠杆菌细胞(LMC500菌株)在28-42℃下在培养基中生长,并如下固定在2.8%FA和0.04%GA中:12.2ml培养物,OD 450, 通过加入1ml 37%FA和21μl25%GA的预混合物,在用于生长的水浴中振荡的同时混合0.2(或0.3的OD 600)。 将培养物转移到50ml Greiner离心管中 注意:建议保持OD <600>低于0.3以在富培养基中实现最佳指数生长,并且将OD <450> <0.2低于最低培养基。
    2. 在室温(RT)下保温15分钟,并在室温下以4000xg离心10分钟。
    3. 在1体积PBS(pH 7.2)中洗涤细胞一次。
    4. 重悬于150μlPBS pH 7.2的沉淀,并将细胞转移到500微升Eppendorf管
    5. 通过在4,500×g(7,000rpm)离心5分钟(RT或4℃)沉淀细胞并在150μlPBS(pH7.2)中洗涤两次。 细胞可以在4℃下储存长达一个月。
    6. 所有后续步骤以150μl(也可能较少)进行,并且所有离心步骤在室温或4℃下以4500rpm×7,000rpm离心5分钟。
    7. 孵育细胞在0.1%Triton X-100/PBS pH 7.2在室温下静置45分钟
    8. 在PBS(pH 7.2)中洗涤细胞三次。
    9. 在室温下将细胞在含有100μg/ml溶菌酶和5mM EDTA的PBS(pH7.2)中孵育45分钟(或在细胞壁突变体的情况下为30分钟)。
    10. 在PBS(pH 7.2)中洗涤细胞三次。

  2. 标签程序
    1. 通过将细胞在PBS(pH 7.2)中的0.5%(w/v)封闭剂中静置或摇动在37℃下孵育30分钟来封闭非特异性结合位点。
    2. 与初级抗体孵育(规则为比免疫印迹所需浓度高10倍),在封闭缓冲液中稀释,在37℃下在振荡培养箱中孵育1-2小时(最小培养时间30分钟,在4℃下最大孵育时间过夜取决于 对抗体)
    3. 在含有0.05%(v/v)Tween-20的PBS(pH 7.2)中洗涤细胞三次
    4. 与在37℃下在封闭缓冲液(1:600)中稀释30分钟的第二抗体驴-α-兔-CY3(保证没有与大肠杆菌交叉反应)孵育30分钟。
      注意:将抗体在封闭溶液中以最大速度离心1分钟,以除去染料团块,然后将其加入细胞。
    5. 在150μlPBS(pH 7.2)/0.05%Tween-20中洗涤细胞三次
    6. 在150μlPBS中洗涤细胞一次。
    7. 将细胞重悬在PBS中。
      注意:
      1. 调整体积至细胞量(通常为20微升),即细胞浓度应足够高以进行显微镜分析。
      2. 通过使用与上述相同的程序将血清与具有缺失的基因的菌株孵育,可以非常方便地将大肠杆菌蛋白质与污染性IgG分离。随后,将未结合的IgG用于与野生型菌株的孵育。如果感兴趣的蛋白质是必需的,则血清必须针对纯蛋白质进行亲和纯化。

笔记

固定细菌培养物(通过甲醛/戊二醛或通过乙醇或甲醇),其对于免疫标记过程是必需的,给细胞提供渗透压休克。膜结合或膜相关蛋白或胞质蛋白的定位不受渗透压休​​克的影响。然而,在周质中自由扩散蛋白可以在固定期间被冲击到细胞极点。因此,我们建议通过分析荧光蛋白融合与这些蛋白质的联合生命成像的本地化验证周质自由扩散蛋白质的本地化。

食谱

  1. PBS缓冲液(pH7.2)(每L)
    140mM NaCl 27 mM KCl
    10mM Na 2 HPO 4 sub缓冲液。 2H 2 2mM KH 2 PO 4 sub/
    注意:PBS应始终超级消毒。

致谢

所描述的方案已经用于以下出版物:Blaauwen等人(1999); Aarsman 。 (2005); Potluri et al。(2010); Typas 等人(2010); Banzhaf 等。 (2012); van der Ploeg等人(2013)和Egan等人(2014)。

参考文献

  1. Aarsman,M.E.,Piette,A.,Fraipont,C.,Vinkenvleugel,T.M.,Nguyen-Disteche,M.andde Blaauwen,T。(2005)。 大肠杆菌分裂的成熟分两步进行。 Mol Microbiol 55(6):1631-1645。
  2. Banzhaf,M.,van den Berg van Saparoea,B.,Terrak,M.,Fraipont,C.,Egan,A.,Philippe,J.,Zapun,A.,Breukink,E.,Nguyen-Disteche, ,den Blaauwen,T。和Vollmer,W​​。(2012)。 肽聚糖合酶在细菌细胞延长中具有活性的协同性。 Mol Microbiol 85(1):179-194。
  3. Den Blaauwen,T.,Buddelmeijer,N.,Aarsman,M.E.,Hameete,C.M。和Nanninga,N。(1999)。 FtsZ大会在大肠杆菌中的定时。 J Bacteriol 181(17):5167-5175。
  4. Egan,AJ,Jean,NL,Koumoutsi,A.,Bougault,CM,Biboy,J.,Sassine,J.,Solovyova,AS,Breukink,E.,Typas,A.,Vollmer,W​​。和Simorre, 2014)。 外膜脂蛋白LpoB跨越周质以刺激肽聚糖合酶PBP1B 。 Proc Natl Acad Sci U S A 111(22):8197-8202。
  5. Hocking,J.,Priyadarshini,R.,Takacs,C.N.,Costa,T.,Dye,N.A。,Shapiro,L.,Vollmer,W​​。和Jacobs-Wagner,C。 重量克分子渗透浓度依赖性将青霉素结合蛋白PBP2转移到新月柄杆菌中的分裂位点。 J Bacteriol 194(12):3116-3127。
  6. Potluri,L.,Karczmarek,A.,Verheul,J.,Piette,A.,Wilkin,JM,Werth,N.,Banzhaf,M.,Vollmer,W​​.,Young,KD,Nguyen-Disteche, den Blaauwen,T。(2010)。 PBP5的腹膜和侧壁定位,大肠杆菌的主要D,D-羧肽酶,需要底物识别和膜附着。 Mol Microbiol 77(2):300-323。
  7. Typas,A.,Banzhaf,M.,van den Berg van Saparoea,B.,Verheul,J.,Biboy,J.,Nichols,RJ,Zietek,M.,Beilharz,K.,Kannenberg,K.,von Rechenberg ,M.,Breukink,E.,den Blaauwen,T.,Gross,CA和Vollmer,W​​。(2010)。 外膜蛋白对肽聚糖合成的调节细胞 143(7):1097-1109。
  8. van der Ploeg,R.,Verheul,J.,Vischer,N.O.,Alexeeva,S.,Hoogendoorn,E.,Postma,M.,Banzhaf,M.,Vollmer,W​​.andde Blaauwen, 在大肠杆菌中的制备性细胞分裂阶段期间细长体和分裂体之间的共定位和相互作用。/em> Mol Microbiol 87(5):1074-1087。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Buddelmeijer, N., Aarsman, M. and Blaauwen, T. d. (2013). Immunolabeling of Proteins in situ in Escherichia coli K12 Strains. Bio-protocol 3(15): e852. DOI: 10.21769/BioProtoc.852.
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