搜索

Co-sedimentation Assay for the Detection of Direct Binding to F-actin
共沉降法检测纤维状肌动蛋白的直接结合   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

This protocol describes measurement of direct protein-protein interactions by actin co-sedimentation assay. The actin co-sedimentation assay is a well-established technique and has been commonly used to demonstrate binding of proteins that interact directly with actin filaments (Ahrens et al., 2012; Mehta and Sibley, 2010; Schuler et al., 2005; Singh et al., 2011). We and others have previously shown that the damaged cell-recognition molecule C-type lectin 9A (Clec9A) recognises a conserved component within nucleated and non-nucleated cells that is exposed only when the cell membrane is damaged (Srivastava and Barber, 2008; Zhang et al., 2012). Here we use Clec9A as an example and present a detailed procedure for demonstrating the direct binding of purified recombinant Clec9A ectodomain to actin filaments.

Keywords: Actin(肌动蛋白), F-actin(F-肌动蛋白), Dendritic Cells(树突状细胞), Clec(中国学习者英语语料库)

Materials and Reagents

  1. Rabbit muscle G-actin (Cytoskeleton, catalog number: AKL95-C )
  2. Recombinant Clec9A ectodomain was expressed and purified by affinity chromatography and size-exclusion chromatography as described previously 5
  3. Tris
  4. CaCl2
  5. ATP
  6. β-mercaptoethanol
  7. KCl
  8. MgCl2
  9. NuPAGE Novex 4-12% Bis-Tris gel (Life Technologies, Invitrogen™, catalog number: NP0327BOX )
  10. Laemmli reducing SDS-PAGE sample buffer (Laemmli 2x concentrate) (Sigma-Aldrich, catalog number: S3401 )
  11. Precision Plus Protein molecular weight marker (Bio-Rad Laboratories, catalog number: 161-0373 )
  12. Coomassie Brilliant Blue
  13. 1x G-buffer (see Recipes)
  14. 10x F-buffer (see Recipes)

Equipment

  1. Superdex S200 10/300 GL column (GE Healthcare)
  2. AKTA Fast Protein Liquid Chromatography (FPLC) system (GE Healthcare)
  3. TLA 100 rotor and Beckman Coulter Optima TL Ultracentrifuge (Beckman Coulter)
  4. Polycarbonate centrifuge tubes (7 x 20 mm, 230 μl) (Beckman Coulter, catalog number: 343775 )
  5. GS-800 calibrated densitometer and Quantity one image analysis software (Transmission densitometer) (Bio-Rad Laboratories, catalog number: 1707980 )

Procedure

  1. Preparation of actin
    1. Reconstitute vial of lyophilised powder of rabbit muscle G-actin in water (2 mg/ml) and incubate protein for 1/2 h at room temperature (RT).
      Note: If making up a large stock of actin, freeze in small, single use aliquots at -80 °C.
    2. Pre-equilibrate a Superdex S200 10/300 GL column with a minimum of 35 ml of G-buffer.
      Note: G-buffer is used as both pre-elution and elution buffer.
    3. Load 0.5 ml of actin (2 mg/ml) onto the Superdex S200, and run column in G-buffer at a flow rate of 0.5 ml/min, and collect column fractions corresponding to elution volumes of 12-15 ml for analysis.
    4. Analyse fractions reflecting the peak of actin protein (G-actin molecular mass is 42-43 kDa) by SDS-PAGE on a NuPAGE Novex 4-12% Bis-Tris gel under reducing conditions, and visualise proteins by Coomassie Brilliant Blue staining to confirm purity of G-actin.
    5. Pool fractions reflecting the peak of G-actin.
    6. Polymerise G-actin (pooled monomeric G-actin from step 1e) to F-actin by adding 1/10 volume of 10x F-buffer and incubating for 1 h at RT.

  2. Assay for actin filament binding
    1. Use the actin filaments generated in step A-6 to set up a series of reaction tubes in polycarbonate ultracentrifuge tubes with a range of concentrations of actin filaments (0, 1, 2, 5 μM) and add a fixed amount of potential actin-interacting protein (Clec9A; 5 μM) or a control protein that does not interact with actin (5 μM) in a total volume of 50 μl. As a control, also include 1 tube of actin alone.
      Note: The concentration of actin filaments used for each reactions should be based on the initial concentration of G-actin in step A-5.
    2. Incubate reactions for 1 h at RT.
    3. Sediment actin filaments and associated proteins by ultracentrifugation at 100,000 x g using a fixed angle rotor for 1 h at RT.
    4. Transfer supernatant to a fresh tube.
    5. Wash pellet by adding 50 μl of 1x F-buffer (diluted in G-buffer) and sediment actin filaments and associated proteins by ultracentrifugation at 100,000 x g for 1 h at RT.
    6. Remove supernatant and resuspend pellet in 1x Laemmli reducing SDS-PAGE sample buffer (equal volume to supernatant).
    7. Set up tubes containing an equal volume of supernatant (from step B-4) with SDS-PAGE sample buffer, or resuspended pellet in SDS-PAGE sample buffer (from step B-6) for each of the reactions of actin +/- actin binding proteins, then incubate samples at 95 °C for 5 min.
    8. Analyse samples by SDS-PAGE and visualise proteins by Coomassie Brilliant Blue staining.
    9. Quantitate the amount of Clec9A in the supernatant and pellet fractions by densitometry analysis using a GS-800 calibrated densitometer.
      Note: It is important to note that as the samples are being analysed under reducing conditions, the sizes of the bands do not change on SDS-PAGE. What changes is the shift of the bands from supernatant to pellet as a result of F-actin cosedimentation.
    10. Calculate the intensity of the Clec9A bands in the pellet, and the intensity of the total amount of Clec9A (pellet + supernatant). Then calculate the proportion of Clec9A co-sedimenting with F-actin as the ratio of Clec9A in the pellet relative to the total Clec9A (Clec9A in the Supernatant + Pellet), that is, the intensity of Clec9A in pellet divided by the intensity of Clec9A in (supernatant + pellet). The proportion of Clec9A co-sedimenting with F-actin relative to a nonspecific binding control is interpreted as the specific binding to F-actin.

Recipes

  1. 1x G-buffer
    20 mM Tris (pH 8)
    0.1 mM CaCl2
    0.1 mM ATP
    5 mM β-mercaptoethanol
  2. 10x F-buffer
    1 M KCl
    20 mM ATP
    20 mM MgCl2

Acknowledgments

Jian-Guo Zhang was supported by Australian Government NHMRC Program Grants 461219 and 1016647. His work was also made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS.

References

  1. Ahrens, S., Zelenay, S., Sancho, D., Hanc, P., Kjaer, S., Feest, C., Fletcher, G., Durkin, C., Postigo, A., Skehel, M., Batista, F., Thompson, B., Way, M., Reis e Sousa, C. and Schulz, O. (2012). F-actin is an evolutionarily conserved damage-associated molecular pattern recognized by DNGR-1, a receptor for dead cells. Immunity 36(4): 635-645.
  2. Mehta, S. and Sibley, L. D. (2010). Toxoplasma gondii actin depolymerizing factor acts primarily to sequester G-actin. J Biol Chem 285(9): 6835-6847.
  3. Schuler, H., Mueller, A. K. and Matuschewski, K. (2005). A Plasmodium actin-depolymerizing factor that binds exclusively to actin monomers. Mol Biol Cell 16(9): 4013-4023.
  4. Singh, B. K., Sattler, J. M., Chatterjee, M., Huttu, J., Schuler, H. and Kursula, I. (2011). Crystal structures explain functional differences in the two actin depolymerization factors of the malaria parasite. J Biol Chem 286(32): 28256-28264.
  5. Srivastava, J. and Barber, D. (2008). Actin co-sedimentation assay; for the analysis of protein binding to F-actin. J Vis Exp(13).
  6. Zhang, J. G., Czabotar, P. E., Policheni, A. N., Caminschi, I., Wan, S. S., Kitsoulis, S., Tullett, K. M., Robin, A. Y., Brammananth, R., van Delft, M. F., Lu, J., O'Reilly, L. A., Josefsson, E. C., Kile, B. T., Chin, W. J., Mintern, J. D., Olshina, M. A., Wong, W., Baum, J., Wright, M. D., Huang, D. C., Mohandas, N., Coppel, R. L., Colman, P. M., Nicola, N. A., Shortman, K. and Lahoud, M. H. (2012). The dendritic cell receptor Clec9A binds damaged cells via exposed actin filaments. Immunity 36(4): 646-657.

简介

This protocol describes measurement of direct protein-protein interactions by actin co-sedimentation assay. The actin co-sedimentation assay is a well-established technique and has been commonly used to demonstrate binding of proteins that interact directly with actin filaments (Ahrens et al., 2012; Mehta and Sibley, 2010; Schuler et al., 2005; Singh et al., 2011). We and others have previously shown that the damaged cell-recognition molecule C-type lectin 9A (Clec9A) recognises a conserved component within nucleated and non-nucleated cells that is exposed only when the cell membrane is damaged (Srivastava and Barber, 2008; Zhang et al., 2012). Here we use Clec9A as an example and present a detailed procedure for demonstrating the direct binding of purified recombinant Clec9A ectodomain to actin filaments.

关键字:肌动蛋白, F-肌动蛋白, 树突状细胞, 中国学习者英语语料库

材料和试剂

  1. 兔肌肉G-肌动蛋白(Cytoskeleton,目录号:AKL95-C)
  2. 重组Clec9A胞外域通过亲和层析和大小排阻层析如前所述5表达和纯化。
  3. Tris
  4. CaCl <2>
  5. ATP
  6. β-巯基乙醇
  7. KCl
  8. MgCl 2
  9. NuPAGE Novex 4-12%Bis-Tris凝胶(Life Technologies,Invitrogen TM,目录号:NP0327BOX)
  10. Laemmli还原SDS-PAGE样品缓冲液(Laemmli 2x浓缩物)(Sigma-Aldrich,目录号:S3401)
  11. Precision Plus蛋白分子量标记(Bio-Rad Laboratories,目录号:161-0373)
  12. 考马斯亮蓝色
  13. 1x G缓冲区(请参阅配方)
  14. 10x F缓冲区(请参阅配方)

设备

  1. Superdex S200 10/300GL柱(GE Healthcare)
  2. AKTA快速蛋白质液相色谱(FPLC)系统(GE Healthcare)
  3. TLA 100转子和Beckman Coulter Optima TL超速离心机(Beckman Coulter)
  4. 聚碳酸酯离心管(7×20mm,230μl)(Beckman Coulter,目录号:343775)
  5. GS-800校准密度计和Quantity一图像分析软件(透射光密度计)(Bio-Rad Laboratories,目录号:1707980)

程序

  1. 肌动蛋白的制备
    1. 重构小瓶的兔肌肉G-肌动蛋白水溶液(2mg/ml)的冻干粉末,并在室温(RT)孵育蛋白质1/2小时。
      注意:如果组成大量的肌动蛋白,将小型单次使用的等分试样冷冻在-80°C。
    2. 用至少35ml的G缓冲液预平衡Superdex S200 10/300 GL柱。
      注意:G-buffer既用作预洗脱,也用作洗脱缓冲液。
    3. 加载0.5毫升肌动蛋白(2毫克/毫升)到Superdex S200,并在G缓冲液中以0.5毫升/分钟的流速运行柱子,收集对应于12-15毫升的洗脱体积的柱级分用于分析。
    4. 通过在还原条件下的NuPAGE Novex 4-12%Bis-Tris凝胶上的SDS-PAGE分析反映肌动蛋白峰(G-肌动蛋白分子量为42-43kDa)的级分,并通过考马斯亮蓝染色观察蛋白质以确认纯度的G-肌动蛋白
    5. 反映G-肌动蛋白峰的泳池部分。
    6. 通过加入1/10体积的10×F-缓冲液并在室温下孵育1小时,将G-肌动蛋白(来自步骤1e的合并的单体G-肌动蛋白)聚合成F-肌动蛋白。

  2. 测定肌动蛋白丝结合
    1. 使用在步骤A-6中产生的肌动蛋白丝在一系列浓度的肌动蛋白丝(0,1,2,5μM)的聚碳酸酯超速离心管中建立一系列反应管,并添加固定量的潜在的肌动蛋白相互作用 蛋白质(Clec9A;5μM)或不与肌动蛋白(5μM)相互作用的对照蛋白质,总体积为50μl。 作为对照,还包括单独的1管肌动蛋白。
      注意:用于每个反应的肌动蛋白丝的浓度应基于步骤A-5中G-肌动蛋白的初始浓度。
    2. 在室温下孵育反应1小时
    3. 通过使用固定角转子在RT下超速离心100,000小时沉淀肌动蛋白丝和相关蛋白1小时。
    4. 将上清液转移到新管中
    5. 通过加入50μl1×F缓冲液(稀释于G缓冲液中)并沉淀肌动蛋白丝和相关蛋白质,通过在100,000xg下超速离心1小时在RT下洗涤沉淀物。
    6. 去除上清液,并在1×Laemmli还原SDS-PAGE样品缓冲液(与上清液等体积)中重悬沉淀
    7. 对于肌动蛋白+/-肌动蛋白的每个反应,设置含有等体积的上清液(来自步骤B-4)的管,用SDS-PAGE样品缓冲液或重悬浮的沉淀在SDS-PAGE样品缓冲液(来自步骤B-6)结合蛋白,然后在95℃孵育样品5分钟
    8. 通过SDS-PAGE分析样品,通过考马斯亮蓝染色观察蛋白质
    9. 通过使用GS-800校准密度计的光密度测定分析定量上清液和沉淀部分中的Clec9A的量。
      注意:重要的是要注意,当样品在还原条件下进行分析时,条带的大小在SDS-PAGE上不改变。作为F-肌动蛋白沉积的结果,从上清液到沉淀物的条带的变化有什么变化。
    10. 计算沉淀中Clec9A条带的强度,以及Clec9A(沉淀+上清液)总量的强度。 然后计算与F-肌动蛋白共沉淀的Clec9A的比例作为沉淀中Clec9A相对于总Clec9A(上清液+沉淀中的Clec9A)的比例,即沉淀中Clec9A的强度除以Clec9A的强度 (上清液+沉淀)。 Clec9A与F-肌动蛋白共沉淀的比例相对于非特异性结合对照被解释为与F-肌动蛋白的特异性结合。

食谱

  1. 1x G缓冲区
    20mM Tris(pH8)
    0.1mM CaCl 2/v/v 0.1 mM ATP
    5mMβ-巯基乙醇
  2. 10x F缓冲区
    1 M KCl
    20 mM ATP
    20mM MgCl 2/

致谢

张建国获澳大利亚政府NHMRC计划拨款461219和1016647支持。他的工作还通过维多利亚州政府运营基础设施支持和澳大利亚政府NHMRC IRIISS实现。

参考文献

  1. Ahrens,S.,Zelenay,S.,Sancho,D.,Hanc,P.,Kjaer,S.,Feest, Fletcher,G.,Durkin,C.,Postigo,A.,Skehel,M.,Batista, Thompson,B.,Way,M.,Reis e Sousa,C。和Schulz,O。(2012)。 F-肌动蛋白是由DNGR-1识别的进化上保守的损伤相关分子模式,受体死细胞。 免疫 36(4):635-645。
  2. Mehta,S。和Sibley,L.D。(2010)。 弓形体弓形体肌动蛋白解聚因子主要用于隔离G-肌动蛋白。 Biol Chem 285(9):6835-6847。
  3. Schuler,H.,Mueller,A.K.and Matuschewski,K。(2005)。 疟原虫肌动蛋白解聚因子,仅与肌动蛋白单体结合。 Biol Cell 16(9):4013-4023。
  4. Singh,B.K.,Sattler,J.M.,Chatterjee,M.,Huttu,J.,Schuler,H。和Kursula,I。(2011)。 晶体结构解释了疟原虫两种肌动蛋白解聚因子的功能差异。 em> J Biol Chem 286(32):28256-28264。
  5. Srivastava,J。和Barber,D。(2008)。 肌动蛋白共沉淀测定; 用于分析蛋白质与F-肌动蛋白的结合。

    (13)。
  6. Zhang,JG,Czabotar,PE,Policheni,AN,Caminschi,I.,Wan,SS,Kitsoulis,S.,Tullett,KM,Robin,AY,Brammananth,R.,van Delft,MF,Lu, 这些研究结果表明,这些研究结果表明,该方法能够有效地降低患者的生活质量,提高患者的生活质量, RL,Colman,PM,Nicola,NA,Shortman,K。和Lahoud,MH(2012)。 树突状细胞受体Clec9A通过暴露的肌动蛋白丝结合受损细胞。免疫 36(4):646-657。
  • 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:Wong, W., Zhang, J., Baum, J., Lahoud, M. H. and Shortman, K. (2012). Co-sedimentation Assay for the Detection of Direct Binding to F-actin. Bio-protocol 2(19): e270. DOI: 10.21769/BioProtoc.270.
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google 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。