搜索

Biotinylation and Purification of Plasma Membrane-associated Proteins from Rodent Cultured Neurons
啮齿动物体外培养神经元的细胞膜蛋白质的生物素化和纯化   

下载 PDF 引用 收藏 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
The Journal of Neuroscience
Jun 2015

Abstract

This protocol aims at the biotin labeling and affinity purification of plasma membrane proteins from cultured neurons. Protein biotinylation consists in the covalent attachment of biotin to proteins. Biotin is a membrane unpermeable molecule with a small size (MW 244.31 g/mol) and therefore does not interfere with the normal function of proteins. Biotin binds to streptavidin and avidin molecules with high affinity. This binding is extremely resistant to temperature, pH and proteolysis, which allows capture and purification of plasma membrane proteins. Moreover, proteins can bind several biotin molecules, that will allow the consequent binding of several streptavidin or avidin molecules, increasing the sensitivity of detection of the proteins of interest. In this protocol proteins at the cell surface of live cultured neurons are biotinylated. Neuronal extracts are prepared and biotinylated proteins are collected with NeutrAvidin-coupled beads, and analyzed by Western blotting.

Keywords: Plasma-membrane proteins (质膜蛋白质), Affinity purification (亲和纯化), Biotin (生物素), Cell surface (细胞表面), Cultured neurons (培养的神经元)

Materials and Reagents

  1. Centrifuge microtube with 0.45 μm filter (VWR International, catalog number: 82031-360 )
  2. Cell scraper with 1.7 cm blade length (SARSTEDT AG & Co, catalog number: 831830 )
  3. Primary cultures of rodent hippocampal or cortical neurons (as described in Caldeira et al., 2007a; Caldeira et al., 2007b; Ferreira et al., 2015)
  4. Glycine (Sigma-Aldrich, catalog number: 15527 )
  5. NeutrAvidin plus ultralink resin (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 53151 )
  6. EZ link sulfo-nhs-ss-biotin (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 21331 )
  7. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626-1 g )
  8. Chymostatin (Sigma-Aldrich, catalog number: C7268-1 mg )
  9. Leupeptin (Sigma-Aldrich, catalog number: L2884-1 mg )
  10. Antipain dihydrochloride from microbial source (Sigma-Aldrich, catalog number: A6191-1 mg )
  11. Pepstatin A (Sigma-Aldrich, catalog number: P5318-5 mg )
  12. Deoxycholic acid (DOC) (Sigma-Aldrich, catalog number: D5670 )
    Note: it is also named “Sodium deoxycholate monohydrate” on Sigma-Aldrich website.
  13. Dithiothreitol (DTT) (NZYTech, catalog number: MB03101 )
  14. Liquid Nitrogen
  15. DMSO (Sigma-Aldrich, catalog number: D8418-100 ml )
  16. PierceTM Bicinchoninic acid protein assay-Reagent A (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23223 )
  17. PierceTM Bicinchoninic acid protein assay-Reagent B (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23224 )
  18. NaCl
  19. KCl
  20. KH2PO4
  21. Na2HPO4
  22. MgCl2
  23. CaCl2
  24. Tris-HCl
  25. EGTA
  26. EDTA
  27. Triton X-100
  28. SDS
  29. Sodium orthovanadate
  30. Bromophenol blue
  31. PBS (see Recipes)
  32. PBS/Ca2+/Mg2+ (see Recipes)
  33. Radioimmune precipitation assay (RIPA) buffer (see Recipes)
  34. Lysis buffer for insoluble proteins (see Recipes)
  35. 2x denaturating buffer (see Recipes)
  36. Protease inhibitor cocktail (see Recipes)

Equipment

  1. Orbital horizontal shaker (GFL, model: 3005 )
  2. Sonicator (Sonics & Materials, model: VC50 )
  3. Benchtop microcentrifuge (Eppendorf AG, model: 5415D )
  4. Laboratory tube rotator (Falc Intruments s.r.l., model: F205 )
  5. Heating block (Bibby Scientific, Stuart Scientific, model: SHT200D )

Procedure

  1. Wash cultured cells (primary cultures of rat or mouse hippocampal or cortical neurons at 91.6 x 103 cells/cm2) twice at 4 °C with PBS/Ca2+/Mg2+ (3 ml per 60 mm Petri dishes or 2 ml per well of a 6-multiwell plate), in non-sterile conditions.
  2. Incubate cells with PBS/Ca2+/Mg2+ containing 0.3 to 1 mg/ml EZ Link Sulfo-NHS-SS-Biotin (adjust accordingly to the specificity of your signal on the biotinylated fraction at the final Western blot) for 45 min at 4 °C under mild shaking on an orbital horizontal shaker, in the dark (2 ml per 60 mm Petri dishes or 1.5 ml per well of a 6 multiwell plate).
  3. Remove the non-bound biotin by washing the cells twice with PBS/Ca2+/Mg2+ supplemented with 100 mM glycine, followed by a 45 min incubation in this solution at 4 ℃ (3 ml per 60 mm Petri dishes or 2 ml per well of a 6-multiwell plate).
  4. Cells lysis:
    1. For low solubility proteins (cells plated in 60 mm Petri dishes): Use 200 μl of lysis buffer supplemented with protease inhibitor cocktail (1:1,000 diluted) per dish, followed by a 30 min incubation on ice. Scrape the cells and transfer the cell extract to a tube. Briefly sonicate (30 sec on ice). Cellular extracts are then incubated with 1% DOC, pH 9.0, 1 h at 37 °C, centrifuged at 18,000 x g for 30 min at 4 °C, and the pellet is discarded.
    2. For soluble proteins (cells plated in 6-multiwell plate): use 400 μl RIPA buffer per well, supplemented with protease inhibitor cocktail (1:1,000 diluted). Scrape the cells and transfer the cell extract to a tube. Briefly freeze the cellular extract in liquid nitrogen. Cellular extracts are thawed and then centrifuged at 6,500 x g, 10 min at 4 °C, and the pellet is discarded.
      Note: Low solubility proteins are difficult to extract with RIPA buffer and the extraction yield is low. Therefore, there is the need to plate higher cell number (thus the use of a 60 mm Petri dish instead of a single well of a 6-multiwell plate), and to optimize the lysis buffer and several protocol steps in order to enhance extraction of low solubility membrane proteins.
  5. Quantification of the protein content in the above supernatants using the bicinchoninic acid (BCA) method. Protein concentration varies according to the initial cell culture density and efficiency of the solubilization; ideally protein concentration range from 0.7 to 1.1 mg/ml should be obtained.
  6. Save 30 μg of protein for total protein blotting.
  7. If more than one sample is analyzed.
    1. For insoluble proteins, dilute the same amount of protein to a final volume of 300 μl of lysis buffer supplemented with 1% DOC.
    2. For soluble proteins, equalize sample concentration and volumes with RIPA buffer supplemented with protease inhibitor cocktail.
  8. To separate the biotinylated proteins, add NeutrAvidin Plus UltraLink Resin in equal amounts to the samples (80 μl for insoluble proteins and 2.5 μl/10 μg of total protein for soluble proteins) and incubate for 2 h at 4 °C with mild shaking on the laboratory tube rotator.
  9. Centrifuge 2,500 x g, for 3 min, discard supernatant and wash the beads three times with 400 μl lysis buffer (lysis buffer for insoluble proteins supplemented with 1% DOC or RIPA buffer for soluble proteins). Last centrifugation may go up to 5 min.
  10. Elute the samples with 2x denaturating buffer at 95 °C for 5 min in a heating block.
  11. Centrifuge the samples at maximum speed into a microtube collector with a 0.45 μm filter. Keep the flow-through which contains the protein fraction. The filter will retain the NeutrAvidin Plus UltraLink Resin.
  12. Run the total amount of total and biotinylated fractions collected in a SDS-PAGE, followed by Western blot using an antibody against the protein of interest. Adjust the thickness and percentage of acrylamide in the gel according to the volume of sample to be loaded, and to the size of the protein of interest. Purity of the biotinylated fraction should be tested by blotting against an intracellular protein like actin or tubulin, which should be absent in the biotinylated fraction.

Recipes

  1. PBS
    137 mM NaCl
    2.7 mM KCl
    1.8 mM KH2PO4
    10 mM Na2HPO4
  2. PBS/Ca2+/Mg2+
    PBS plus 0.5 mM MgCl2 and 1 mM CaCl2 (pH 7.4)
  3. RIPA buffer
    150 mM NaCl
    50 mM Tris-HCl (pH 7.4)
    5 mM EGTA
    1% Triton
    0.5% DOC
    0.1% SDS (pH 7.5)
  4. Lysis buffer
    50 mM Tris-HCl (pH 7.5)
    1 mM DTT
    5 mM EGTA
    10 μM EDTA
  5. 2x denaturating buffer
    125 mM Tris (pH 6.8)
    100 mM glycine
    4% SDS
    200 mM dithiothreitol
    40% glycerol
    3 mM sodium orthovanadate
    0.01% bromphenol blue
  6. Protease inhibitor cocktail
    0.1 M phenylmethylsulfonyl fluoride (in DMSO)
    CLAP (in DMSO):
    1. 1 mg/ml chymostatin
    2. 1 mg/ml leupeptin
    3. 1 mg/ml antipain
    4. 1 mg/ml pepstatin

Acknowledgments

Work in the authors' laboratories was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal, and Fundo Europeu de Desenvolvimento Regional (FEDER and COMPETE).

References

  1. Caldeira, M. V., Melo, C. V., Pereira, D. B., Carvalho, R., Correia, S. S., Backos, D. S., Carvalho, A. L., Esteban, J. A. and Duarte, C. B. (2007a). Brain-derived neurotrophic factor regulates the expression and synaptic delivery of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits in hippocampal neurons. J Biol Chem 282(17): 12619-12628.
  2. Caldeira, M. V., Melo, C. V., Pereira, D. B., Carvalho, R. F., Carvalho, A. L. and Duarte, C. B. (2007b). BDNF regulates the expression and traffic of NMDA receptors in cultured hippocampal neurons. Mol Cell Neurosci 35(2): 208-219.
  3. Ferreira, J. S., Schmidt, J., Rio, P., Águas, R., Rooyakkers, A., Li, K. W., Smit, A. B., Craig, A. M. and Carvalho, A. L. (2015). GluN2B-containing NMDA receptors regulate AMPA receptor traffic through anchoring of the synaptic proteasome. J Neurosci 35(22): 8462-8479.

简介

该方案旨在从培养的神经元生物素标记和亲和纯化质膜蛋白。 蛋白质生物素化包括生物素与蛋白质共价连接。 生物素是具有小尺寸(MW 244.31g / mol)的膜不可渗透分子,因此不干扰蛋白质的正常功能。 生物素以高亲和力结合链霉抗生物素蛋白和抗生物素蛋白分子。 这种结合对温度,pH和蛋白水解具有极高的抗性,可以捕获和纯化质膜蛋白。 此外,蛋白质可以结合几个生物素分子,这将允许几个链霉抗生物素蛋白或抗生物素蛋白分子的结合,增加感兴趣的蛋白质的检测灵敏度。 在该方案中,活的培养神经元的细胞表面的蛋白质被生物素化。 制备神经元提取物并用NeutrAvidin偶联的珠收集生物素化的蛋白质,并通过Western印迹进行分析。

关键字:质膜蛋白质, 亲和纯化, 生物素, 细胞表面, 培养的神经元

材料和试剂

  1. 用0.45μm过滤器(VWR International,目录号:82031-360)离心微量管
  2. 具有1.7cm刀片长度的细胞刮刀(SARSTEDT AG& Co,目录号:831830)
  3. 啮齿动物海马或皮层神经元的原代培养物(如Caldeira等人,2007a; Caldeira等人,2007b; Ferreira等人, 。,2015)
  4. 甘氨酸(Sigma-Aldrich,目录号:15527)
  5. NeutrAvidin plus ultralink resin(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:53151)
  6. EZ链接磺基-nhs-β-生物素(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:21331)
  7. 苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626-1g)
  8. 胰凝乳蛋白酶抑制剂(Sigma-Aldrich,目录号:C7268-1mg)
  9. 亮肽素(Sigma-Aldrich,目录号:L2884-1mg)
  10. 来自微生物来源的二盐酸盐(Sigma-Aldrich,目录号:A6191-1mg)
  11. 胃酶抑素A(Sigma-Aldrich,目录号:P5318-5mg)
  12. 脱氧胆酸(DOC)(Sigma-Aldrich,目录号:D5670)
    注意:在Sigma-Aldrich网站上也称为"脱氧胆酸钠一水合物"。
  13. 二硫苏糖醇(DTT)(NZYTech,目录号:MB03101)
  14. 液氮
  15. DMSO(Sigma-Aldrich,目录号:D8418-100ml)
  16. Pierce公司的双金鸡宁酸蛋白质测定 - 试剂A(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:23223)
  17. Pierce公司双二辛酸蛋白质测定 - 试剂B(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:23224)
  18. NaCl
  19. KCl
  20. KH 2 PO 4
  21. Na HPO 4
  22. MgCl 2
  23. CaCl <2>
  24. Tris-HCl
  25. EGTA
  26. EDTA
  27. Triton X-100
  28. SDS
  29. 原钒酸钠
  30. 溴酚蓝
  31. PBS(请参阅配方)
  32. PBS/Ca 2 + /Mg 2 +
  33. 放射免疫沉淀测定(RIPA)缓冲液(参见配方)
  34. 不溶性蛋白的裂解缓冲液(见配方)
  35. 2x变性缓冲液(见配方)
  36. 蛋白酶抑制剂混合物(见配方)

设备

  1. 轨道水平摇动器(GFL,型号:3005)
  2. 超声波仪(Sonics& Materials,型号:VC50)
  3. 台式微量离心机(Eppendorf AG,型号:5415D)
  4. 实验室管旋转器(Falc Intruments s.r.l.,型号:F205)
  5. 加热块(Bibby Scientific,Stuart Scientific,型号:SHT200D)

程序

  1. 在4℃下用PBS/Ca 2+洗涤培养的细胞(大鼠或小鼠海马或皮质神经元在91.6×10 3细胞/cm 2的原代培养物)两次,在非无菌条件下进行培养。在非无菌条件下,将培养基(每60mm陪替氏培养皿3ml或6孔多孔培养板的2ml /孔)
  2. 将细胞与含有0.3至1mg/ml EZ Link Sulfo-NHS-SS-生物素的PBS/Ca 2+ 2+/Mg 2+ 2 +缓冲液混合(根据您的特异性在黑暗中(2ml/60mm皮氏培养皿或6孔多孔板的每孔1.5ml),在4℃,在轨道水平振荡器上轻微摇动下,在生物素化级分上的信号(在最终蛋白质印迹上)。
  3. 通过用补充有100mM甘氨酸的PBS/Ca 2+ +/Mg 2+洗涤细胞两次,然后在该溶液中孵育45分钟,除去未结合的生物素在4℃(每60mm培养皿3ml或6ml多孔板的每孔2ml)
  4. 细胞裂解:
    1. 对于低溶解度蛋白(细胞接种在60mm培养皿中):使用 补充有蛋白酶抑制剂混合物的200μl裂解缓冲液 (1:1,000稀释),随后在冰上温育30分钟。 刮细胞并将细胞提取物转移到试管中。简要地说 超声(在冰上30秒)。然后将细胞提取物与1% DOC,pH 9.0,在37℃下1小时,在4℃以18,000×g离心30分钟,  并丢弃沉淀。
    2. 对于可溶性蛋白(细胞铺板  在6多孔板中):每孔使用400μlRIPA缓冲液,补充 用蛋白酶抑制剂混合物(1:1000稀释)。刮细胞和  将细胞提取物转移到管中。短暂冷冻细胞 提取液在液氮中。然后将细胞提取物解冻 在6500×g离心,在4℃离心10分钟,弃去沉淀。
      注意:低溶解度蛋白质难以用RIPA缓冲液提取  提取率低。因此,需要镀板更高的细胞数量(因此使用60mm培养皿代替 6多孔板的单孔),并优化裂解缓冲液 和几个协议步骤,以增强低的提取 溶解性膜蛋白
  5. 使用二金鸡宁酸(BCA)方法定量上述上清液中的蛋白质含量。 蛋白质浓度根据初始细胞培养密度和增溶效率而变化; 理想地,应当获得0.7至1.1mg/ml的蛋白质浓度范围
  6. 保存30μg蛋白质用于总蛋白质印迹
  7. 如果分析了多个样品。
    1. 对于不溶性蛋白质,将相同量的蛋白质稀释至最终体积为300μl的补充有1%DOC的裂解缓冲液。
    2. 对于可溶性蛋白质,平衡样品浓度和体积   补充有蛋白酶抑制剂混合物的RIPA缓冲液。
  8. 为了分离生物素化的蛋白质,向样品中加入等量的NeutrAvidin Plus UltraLink Resin(对于不溶性蛋白质为80μl,对于可溶性蛋白质为2.5μl/10μg总蛋白质),并在4℃下温和振荡在2℃孵育2小时 实验室管旋转器。
  9. 离心2,500xg,3分钟,弃去上清液,并用400μl裂解缓冲液(用于溶解蛋白质的裂解缓冲液,用于补充有1%DOC或用于可溶性蛋白质的RIPA缓冲液)洗涤珠子三次。 最后一次离心可能需要5分钟。
  10. 用2×变性缓冲液在95℃下在加热块中洗脱样品5分钟
  11. 以最大速度将样品离心到具有0.45μm过滤器的微管收集器中。 保持含有蛋白质级分的流通液。 过滤器将保留NeutrAvidin Plus UltraLink Resin。
  12. 运行在SDS-PAGE中收集的总量和生物素化级分的总量,然后使用针对目标蛋白的抗体进行Western印迹。 根据要装载的样品的体积,以及感兴趣的蛋白质的大小,调整凝胶中丙烯酰胺的厚度和百分比。 生物素化馏分的纯度应通过对细胞内蛋白质如肌动蛋白或微管蛋白(其在生物素化级分中不存在)进行印迹测试。

食谱

  1. PBS
    137 mM NaCl 2.7 mM KCl
    1.8mM KH 2 PO 4 sub/
    10mM Na 2 HPO 4
  2. PBS/Ca 2 + /Mg 2 +
    PBS加0.5mM MgCl 2和1mM CaCl 2(pH 7.4)的缓冲液中。
  3. RIPA缓冲区
    150mM NaCl 50mM Tris-HCl(pH7.4) 5mM EGTA
    1%Triton
    0.5%DOC
    0.1%SDS(pH7.5)
  4. 裂解缓冲液
    50mM Tris-HCl(pH7.5) 1 mM DTT
    5mM EGTA
    10μMEDTA
  5. 2x变性缓冲液
    125mM Tris(pH6.8) 100mM甘氨酸 4%SDS
    200mM二硫苏糖醇 40%甘油 3 mM原钒酸钠 0.01%溴酚蓝
  6. 蛋白酶抑制剂混合物
    0.1M苯甲基磺酰氟(在DMSO中)
    CLAP(在DMSO中):
    一个。 1mg/ml抑糜蛋白药 b。 1mg/ml亮肽素
    C。 1mg/ml抗蛋白酶
    d。 1mg/ml胃酶抑素

致谢

作者实验室的工作由葡萄牙基金会技术委员会(FCT)和欧洲地区基金会(FEDER和COMPETE)支持。

参考文献

  1. Caldeira,M.V.,Melo,C.V.,Pereira,D.B.,Carvalho,R.,Correia,S.S.,Backos,D.S.,Carvalho,A.L.,Esteban,J.A。和Duarte,C.B。 脑源性神经营养因子调节α-氨基-3-羟基-5-甲基胞嘧啶的表达和突触传递 - 甲基-4-异恶唑丙酸受体亚基在海马神经元中。 J Biol Chem 282(17):12619-12628。
  2. Caldeira,M.V.,Melo,C.V.,Pereira,D.B.,Carvalho,R.F.,Carvalho,A.L.and Duarte,C.B。(2007b)。 BDNF调整的表达和流量NMDA receptors in cultured hippocampal neurons。 Mol Cell Neurosci 35(2):208-219。
  3. Ferreira,J.S.,Schmidt,J.,Rio,P.,Águas,R.,Rooyakkers,A.,Li,K.W.,Smit,A.B.,Craig,A.M.and Carvalho,A.L。 含GluN2B的NMDA受体通过锚定突触蛋白酶体调节AMPA受体通讯。 em> J Neurosci 35(22):8462-8479。
  • 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: © 2016 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. Caldeira, M., Ferreira, J. S., Carvalho, A. L. and Duarte, C. B. (2016). Biotinylation and Purification of Plasma Membrane-associated Proteins from Rodent Cultured Neurons. Bio-protocol 6(10): e1807. DOI: 10.21769/BioProtoc.1807.
  2. Ferreira, J. S., Schmidt, J., Rio, P., Águas, R., Rooyakkers, A., Li, K. W., Smit, A. B., Craig, A. M. and Carvalho, A. L. (2015). GluN2B-containing NMDA receptors regulate AMPA receptor traffic through anchoring of the synaptic proteasome. J Neurosci 35(22): 8462-8479.
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。