搜索

Protein-lipid Interaction Analysis by Surface Plasmon Resonance (SPR)
表面等离子共振 (SPR)法分析蛋白脂质的相互作用   

评审
匿名评审
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

Interactions of lipids with proteins are essential events in the framework of biological membranes. Assessment of the affinity and specificity of protein-lipid binding can give useful information to elucidate cell membrane functions. Surface Plasmon Resonance (SPR) is a powerful technology to study macromolecular interactions, allowing direct and rapid determination of association and dissociation rates using small amounts of samples. An extensive range of binding analyses can be performed by SPR such as protein–protein, protein–membrane (lipids), protein–carbohydrate, protein–nucleic acid and even protein-small molecules. This protocol describes the binding of an antimicrobial protein (used as ligand) to a lipopolysaccharide (LPS) (used as analyte) after immobilization onto a CM sensor chip by amine coupling.

Materials and Reagents

  1. Purified protein as ligand (purity >90%) (1 ml at minimum 20 µg/ml)
  2. Purified Lipid as analyte (minimum 300 µl at 2 mg/ml)
  3. 10 mM acetate buffer (pH 4 to 5.5) (GE Healthcare, catalog numbers: BR-1003-49 to 52)
  4. Filtered deionized water
  5. 200 mM NaOH
  6. HBS-EP buffer (GE Healthcare, catalog number: BR-1001-88 ) (see Recipes)
  7. Amine coupling kit components (GE Healthcare, catalog number: BR-10000-50 ) (see Recipes)
  8. Regeneration scouting kit components (GE Healthcare, catalog number: BR-1005-56 ) (see Recipes)

Equipment

  1. Biacore 3000 system (GE Healthcare)
  2. CM 5 sensor chip with carboxyl groups available for the amine coupling reaction (research grade) (GE Healthcare, catalog number: BR-1003-99 )
  3. Glass vials (9 mm)
  4. Glass vials (16 mm)
  5. 1.5 ml centrifuge tubes
  6. Sonicator (Branson Bath-type ultrasonicator, model: 5510 )

Software

  1. BiaEvaluation software (GE Healthcare)

Procedure

  1. Preparation of reagents
    1. Prepare reagents of the amine coupling kit according to the instruction datasheet (dissolve EDC and NHS in 10 ml filtered deionized water to obtain solutions at 400 mM and 100 mM respectively).
    2. Allow all other reagents (1.0 M ethanolamine-HCl, HBS-EP buffer, acetate buffers and regeneration scouting kit solutions) to warm up to room temperature  (22-25 °C) before use.

  2. pH scouting
    pH scouting allows to determine the optimal pH and ionic strength for ligand immobilization. This step is necessary when working with carboxymethylated dextran matrix (CM) sensors.
    1. Set sensor chip into the Biacore instrument (follow instrument's handbook).
    2. Set the HBS-EP buffer as running buffer.
    3. Set up the immobilization pH scouting function in the Biacore control software.
    4. Prepare 100 µl of ligand solutions by diluting ligand to a final concentration of 20-200 μg/ml in 10 mM acetate buffers at different pH (4.0, 4.5, 5.0 and 5.5).  
    5. Inject 80 µl of each ligand solutions and determine the optimal pH in which the highest pre-concentration response is observed (Figure 1). After each ligand injection the sensor chip surface is washed with running buffer (already set up in the pH scouting function).
    6. At the end of the pH scouting, inject 220 µl of 1 M ethanolamine-HCl. This regeneration step is performed to completely remove the ligand from the sensor chip surface.
      Note: Select your running buffer depending on the type of molecules to interact, which kind of assay will be run, and the type of sensor chip used. In this case, buffers free of primary amine groups or strong nucleophilic groups (e.g. sodium acetate) and of low ionic strength (e.g. 10 mM) are recommended for the electrostatic attraction to occur.
      If two pH conditions give similar pre-concentration results, select the higher pH in order to avoid ligand denaturation or precipitation.


    Figure 1. pH scouting sensorgram A and results report B showing responses generated by the ligand. In this case 4.5 is the optimal pH for ligand immobilization. Y-axial variable illustrates the response in resonance units (RU) after ligand injections and X-axial variable shows the time settled for all injections.

  3. Immobilization of ligand
    1. Open the immobilization application in the Biacore control software and set up your template for immobilization.
    2. Prepare the ligand in the acetate buffer with the pH you chose in step B.
    3. Prepare vials with EDC, NHS and ethanolamine-HCl solutions. The volume of ligand and solutions (EDC, NHS and ethanolamine-HCl) required is displayed in the immobilization application and depends on how many flow cells will be used (Figure 2).
    4. Run the immobilization procedure. The application will inject an EDC/NHS mix (1: 1) to activate the chip surface for ligand immobilization, then ligand solution will be injected. Finally, an injection of ethanolamine-HCl will be performed to wash away unbound ligand and deactivate remaining active carboxyl groups.
    5. Check immobilization results (Figure 3), evaluate the response in terms of analyte binding capacity of the surface and not on the response units observed. Optimal immobilization level depends on the relative molecular weights of the ligand and analyte. For this protocol, ligand immobilization was fixed to 7,000 RU. Binding capacity depends on the molecular weight of the analyte in relation to the number of ligand sites on the sensor chip surface. Binding capacity for ligand shown in Figure 3 was ~118 RU.  
      Note: If you are going to use the same analyte for several ligands, you can immobilize them all before continuing with step C4. At this point, you can stop the experiment and store the sensor chip at 4 °C.


    Figure 2. Example of immobilization template showing volume solutions necessary to run the immobilization procedure


    Figure 3. Sensorgram A and result report B of ligand immobilization. Response 1 shows the amount of ligand bound to the surface after injection and response 2 illustrates the final amount of ligand covalently immobilized after surface deactivation.

  4. Analyte preparation
    1. Sonicate (at 40 kHz) stock solution of LPS or lipid A for 15 min at 25 °C. Since LPS tends to aggregate, it is important to sonicate LPS and lipid A solutions just before preparing analyte samples.
    2. Prepare analyte at the desired concentrations in HBS-EP buffer. In our case, LPS was diluted at 5, 10, 25, 50, 100 and 200 µg/ml and lipid A at 3, 6, 10, 15, 20, 30, 40, 50 µg/ml.

  5. Regeneration conditions
    Regeneration scouting allows to determine optimal conditions to remove bound analyte from the ligand on the sensor chip surface. This step is important for a successful binding assay; it allows to identify the appropriate regeneration solution that will completely remove the analyte without destroying ligand activity. Regeneration conditions need to be determined for each analyte.
    1. Open the regeneration scouting application in the Biacore control software.
    2. Inject the highest analyte concentration (e.g. 200 µg/ml).
    3. Test the buffers, starting with the mildest. First inject glycine buffers at pH 3.0, 2.5, 2.0 and 1.5 for 60 sec. If the response level does not go back to the baseline level (response before the analyte injection), keep on testing other buffers. Optimization of contact time of the surface with the regeneration buffer, number of buffer injections and baseline stabilization period will help to determine optimal regeneration conditions. In our case regeneration was set up with two washes of 20 mM NaOH for 5 min for LPS and 150 mM NaOH for 5 min for lipid A, followed by 2 min of wash with HBS-EP buffer (Figure 4).
    4. Once you have identified good regeneration conditions for your experiment, perform several cycles of analyte injection and regeneration in order to evaluate ligand performance (responses obtained from the binding assays should not vary more than 10% of the response of the first injection).  
      Note: The regeneration scouting kit provides several ready-to use buffers with a range of pH values and different ionic strength but alternative home-made buffers can also be used as long as they are filtered and degassed.


    Figure 4. Regeneration scouting sensorgram A and result report B illustrating some conditions tested for analyte (lipid A) removal

  6. Binding assay
    Before starting your binding assay, design your experimental plan taking into consideration:
    1. Flow rate and injection time for the antigen solution: need to be optimized according to your ligand. Bear in mind that high flow rates help to avoid any mass transport effects and that long enough injections (and wash) allow to observe a curvature of the binding response and a decay in the dissociation phase.
    2. Washing conditions: Use the previously optimized regeneration conditions.  
    3. Blank control: An activated and blocked flow-cell without immobilized ligand is necessary as a reference to evaluate nonspecific binding.
    4. Negative control: Additionally to the blank, it is recommended to use a non-relevant protein of similar size of your ligand as a negative control of binding. This second control is helpful to validate your experiment since the stickiness of lipids may give false binding results.
      Once you have established your experimental conditions, proceed to perform your binding assay, you can use the binding analysis application provided with Biacore control software or customize your own application and templates following instrument’s manual. Typical results obtained in a lipid binding assay are exemplified in Figure 5.
      Note: If using a new chip perform 3-5 cycles of binding before starting your real experiment to verify whether ligand-analyte interaction response is steady.


    Figure 5. Sensorgram of protein-lipid (LPS) binding assay. In this example, binding experiment was performed at the same time with three different proteins.

  7. Binding analysis
    Collected data are then analyzed using the BIAevaluation software (follow software handbook instructions - GE Healthcare). Figure 6 shows an example of results obtained after analysis of a protein-lipid binding assay. Experiments done in triplicate gave similar results.


    Figure 6. Final sensorgram of protein-lipid (LPS) interaction

Notes

  1. Due to the inherent stickiness of lipids, it is strongly advised to thoroughly clean the instrument (desorb and sanitize) after binding experiments to remove traces of analyte remaining in the circuits of the Biacore system.
  2. If several ligands and analytes will be analyzed by SPR, it is recommended to have at least two sensor chips. Foreseeing a sensor chip stock is useful in case one or more ligands are damaged when adjusting regeneration conditions.

Recipes

  1. HBS-EP buffer
    0.01 M HEPES (pH 7.4)
    0.15 M NaCl
    3 mM EDTA
    0.005% (v/v) P20 Surfactant
  2. Amine coupling kit components
    750 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)
    115 mg N-hydroxysuccinimide (NHS)
    10.5 ml 1.0 M ethanolamine-HCl (pH 8.5)
  3. Regeneration scouting kit components
    11 ml ethylene glycol (p.a.)
    11 ml 10 mM glycine-HCl (pH 1.5)
    11 ml 10 mM glycine-HCl (pH 2.0)
    11 ml 10 mM glycine-HCl (pH 2.5)
    11 ml 10 mM glycine-HCl (pH 3.0)
    11 ml 4.0 M magnesium chloride
    11 ml 0.2 M sodium hydroxide
    11 ml 0.5% sodium dodecyl sulphate (SDS)
    11 ml 5.0 M sodium chloride
    20 ml surfactant P20

Acknowledgments

The authors wish to thank the Analytical Biochemistry Facility of the Sophia Agrobiotech Institute (ISA) for kind access to the Biacore system. This work was funded by ANR (ANR-07-BLAN-0214 and ANR-12-EMMA-00O7-01), CNRS and INRA.

References

  1. Baron, O. L., van West, P., Industri, B., Ponchet, M., Dubreuil, G., Gourbal, B., Reichhart, J. M. and Coustau, C. (2013). Parental transfer of the antimicrobial protein LBP/BPI protects Biomphalaria glabrata eggs against oomycete infections. PLoS Pathog 9(12): e1003792.

简介

脂质与蛋白质的相互作用是生物膜框架中的重要事件。 蛋白质 - 脂质结合的亲和力和特异性的评估可以提供有用的信息来阐明细胞膜功能。 表面等离子体共振(SPR)是研究大分子相互作用的强大技术,允许使用少量样品直接和快速测定缔合和解离速率。 可以通过SPR进行广泛范围的结合分析,例如蛋白质 - 蛋白质,蛋白质 - 膜(脂质),蛋白质 - 碳水化合物,蛋白质 - 核酸甚至蛋白质 - 小分子。 该方案描述了在通过胺偶联固定在CM传感器芯片上之后,抗微生物蛋白(用作配体)与脂多糖(LPS)(用作分析物)的结合。

材料和试剂

  1. 纯化的蛋白质作为配体(纯度> 90%)(1ml,至少20μg/ml)
  2. 纯化的脂质作为分析物(最小300μl,2mg/ml)
  3. 10mM乙酸缓冲液(pH4〜5.5)(GE Healthcare,目录号:BR-1003-49〜52)
  4. 过滤的去离子水
  5. 200 mM NaOH
  6. HBS-EP缓冲液(GE Healthcare,目录号:BR-1001-88)(参见Recipes)
  7. 胺偶联试剂盒组分(GE Healthcare,目录号:BR-10000-50)(参见Recipes)
  8. 再生侦查套件组件(GE Healthcare,目录号:BR-1005-56)(参见配方)

设备

  1. Biacore 3000系统(GE Healthcare)
  2. 可用于胺偶联反应的羧基的CM 5传感器芯片(研究级)(GE Healthcare,目录号:BR-1003-99)
  3. 玻璃小瓶(9毫米)
  4. 玻璃小瓶(16 mm)
  5. 1.5ml离心管
  6. 超声波仪(Branson Bath型超声波发生器,型号:5510)

软件

  1. BiaEvaluation软件(GE Healthcare)

程序

  1. 试剂的制备
    1. 根据说明书数据表制备胺偶联试剂盒的试剂(将EDC和NHS溶解在10ml过滤的去离子水中,分别得到400mM和100mM的溶液)。
    2. 使所有其他试剂(1.0M乙醇胺-HCl,HBS-EP缓冲液,乙酸盐缓冲液和再生侦测试剂盒溶液)温热至室温, (22-25℃)。

  2. pH检测
    pH侦测允许确定用于配体固定的最佳pH和离子强度。 当使用羧甲基化葡聚糖基质(CM)传感器时,该步骤是必需的。
    1. 将传感器芯片装入Biacore仪器(按照仪器的手册)。
    2. 将HBS-EP缓冲区设置为运行缓冲区。
    3. 在Biacore控制软件中设置固定pH侦测功能。
    4. 通过在不同pH(4.0,4.5,5.0和5.5)的10mM乙酸盐缓冲液中稀释配体至最终浓度为20-200μg/ml,制备100μl配体溶液。  
    5. 注入80μl的每个配体溶液,并确定最佳的pH,其中观察到最高的预浓度反应(图1)。 在每个配体注射后,用运行缓冲液(已经在pH侦测功能中建立)洗涤传感器芯片表面。
    6. 在pH研究结束时,注射220μl的1M乙醇胺-HCl。执行该再生步骤以从传感器芯片表面完全去除配体 注意:根据要相互作用的分子类型,运行哪种测定法以及使用的传感器芯片类型,选择运行缓冲液。在这种情况下,建议使用不含伯胺基团或强亲核基团(例如乙酸钠)和低离子强度(例如10mM)的缓冲液进行静电吸引。
      如果两个pH条件产生类似的预浓缩结果,则选择较高的pH以避免配体变性或沉淀。


    图1. pH检测传感图A和结果报告B显示由配体产生的响应。在这种情况下,4.5是配体固定的最佳pH。 Y轴变量说明配体注射后共振单位(RU)的响应,X轴变量显示所有注射的时间。

  3. 配体的固定化
    1. 在Biacore控制软件中打开固定应用程序,并设置您的模板用于固定。
    2. 使用您在步骤B中选择的pH,在乙酸盐缓冲液中制备配体
    3. 用EDC,NHS和乙醇胺-HCl溶液制备小瓶。所需的配体和溶液(EDC,NHS和乙醇胺-HCl)的体积显示在固定化应用中,并且取决于将使用多少流动池(图2)。
    4. 运行固定程序。应用程序将注入EDC/NHS混合物(1:1)以激活芯片表面进行配体固定,然后注入配体溶液。最后,注射乙醇胺-HCl以洗去未结合的配体并使剩余的活性羧基失活
    5. 检查固定结果(图3),根据表面的分析物结合能力而不是观察到的响应单位评价响应。最佳固定水平取决于配体和分析物的相对分子量。对于该方案,将配体固定固定为7,000RU。结合能力取决于分析物的分子量与传感器芯片表面上配体位点的数目的关系。图3中显示的配体的结合能力为〜118RU。  
      注意:如果您要对多个配体使用相同的分析物,则可以在继续执行步骤C4之前将其固定。此时,您可以停止实验并将传感器芯片存储在4°C。


    图2.固定模板示例,显示运行固定程序所需的体积解决方案


    图3.传感图A和配体固定的结果报告B. 响应1显示在注射后结合到表面的配体的量,反应2说明在表面失活后共价固定的配体的最终量。 />
  4. 分析物制备
    1. 在25℃下超声处理(40kHz)LPS或脂质A的储备液15分钟。由于LPS倾向于聚集,所以在准备分析物样品之前对LPS和脂质A溶液进行超声处理是重要的
    2. 在HBS-EP缓冲液中制备所需浓度的分析物。在我们的情况下,LPS以5,10,25,50,100和200μg/ml和3,6,10,15,20,30,40,50μg/ml的脂质A稀释。

  5. 再生条件
    再生侦测允许确定从传感器芯片表面上的配体中除去结合的分析物的最佳条件。该步骤对于成功的结合测定是重要的;它允许鉴定将完全去除分析物而不破坏配体活性的合适的再生溶液。需要确定每种分析物的再生条件。
    1. 在Biacore控制软件中打开再生侦查应用程序。
    2. 注射最高分析物浓度(例如 200μg/ml)
    3. 测试缓冲区,从最温和的开始。首先注射pH 3.0,2.5,2.0和1.5的甘氨酸缓冲液60秒。如果反应水平没有回到基线水平(分析物注射前的反应),则继续测试其他缓冲液。优化表面与再生缓冲液的接触时间,缓冲液注射次数和基线稳定期将有助于确定最佳再生条件。在我们的情况下,对于LPS,使用20mM NaOH洗涤5分钟,对于脂质A使用150mM NaOH洗涤5分钟,然后用HBS-EP缓冲液洗涤2分钟(图4)。

    4. 再生条件
      再生侦测允许确定从传感器芯片表面上的配体中除去结合的分析物的最佳条件。该步骤对于成功的结合测定是重要的;它允许鉴定将完全去除分析物而不破坏配体活性的合适的再生溶液。需要确定每种分析物的再生条件。
      1. 在Biacore控制软件中打开再生侦查应用程序。
      2. 注射最高分析物浓度(例如 200μg/ml)
      3. 测试缓冲区,从最温和的开始。首先注射pH 3.0,2.5,2.0和1.5的甘氨酸缓冲液60秒。如果反应水平没有回到基线水平(分析物注射前的反应),则继续测试其他缓冲液。优化表面与再生缓冲液的接触时间,缓冲液注射次数和基线稳定期将有助于确定最佳再生条件。在我们的情况下,对于LPS,使用20mM NaOH洗涤5分钟,对于脂质A使用150mM NaOH洗涤5分钟,然后用HBS-EP缓冲液洗涤2分钟(图4)。
      4. ... Washing conditions: Use the previously optimized regeneration conditions.  
      5. Blank control: An activated and blocked flow-cell without immobilized ligand is necessary as a reference to evaluate nonspecific binding.
      6. Negative control: Additionally to the blank, it is recommended to use a non-relevant protein of similar size of your ligand as a negative control of binding. This second control is helpful to validate your experiment since the stickiness of lipids may give false binding results.
        Once you have established your experimental conditions, proceed to perform your binding assay, you can use the binding analysis application provided with Biacore control software or customize your own application and templates following instrument’s manual. Typical results obtained in a lipid binding assay are exemplified in Figure 5.
        Note: If using a new chip perform 3-5 cycles of binding before starting your real experiment to verify whether ligand-analyte interaction response is steady.


      Figure 5. Sensorgram of protein-lipid (LPS) binding assay. In this example, binding experiment was performed at the same time with three different proteins.

    5. 绑定分析
      然后使用BIA评估软件(遵循软件手册说明书-GE Healthcare)分析收集的数据。图6显示了蛋白质 - 脂质结合测定分析后获得的结果的实例。一式三份进行的实验得到相似的结果。


      图6.蛋白质 - 脂质(LPS)相互作用的最终传感图

    笔记

    1. 由于脂质固有的粘性,强烈建议在结合实验后彻底清洗仪器(解吸和消毒),以清除Biacore系统回路中残留的痕量分析物。
    2. 如果通过SPR分析几种配体和分析物,建议至少有两个传感器芯片。在调节再生条件时,如果一个或多个配体损坏,预见传感器芯片料是有用的

    食谱

    1. HBS-EP缓冲区
      0.01 M HEPES(pH 7.4)
      0.15 M NaCl
      3 mM EDTA
      0.005%(v/v)P20表面活性剂
    2. 胺偶联套件组件
      750mg 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC) 115mg N-羟基琥珀酰亚胺(NHS) 10.5ml 1.0M乙醇胺-HCl(pH8.5)
    3. 再生侦测套件组件
      11 ml乙二醇(p.a.) 11ml 10mM甘氨酸-HCl(pH1.5) 11ml 10mM甘氨酸-HCl(pH2.0) 11ml 10mM甘氨酸-HCl(pH2.5) 11ml 10mM甘氨酸-HCl(pH3.0) 11ml 4.0M氯化镁
      11ml 0.2M氢氧化钠
      11ml 0.5%十二烷基硫酸钠(SDS)
      11ml 5.0M氯化钠
      20ml表面活性剂P20

    致谢

    作者希望感谢索菲亚农业生物技术研究所(ISA)的分析生物化学设施,以便接近Biacore系统。 这项工作由ANR(ANR-07-BLAN-0214和ANR-12-EMMA-00O7-01),CNRS和 INRA。

    参考文献

    1. Baron,O.L.,van West,P.,Industri,B.,Ponchet,M.,Dubreuil,G.,Gourbal,B.,Reichhart,J.M.and Coustau,C。 抗微生物蛋白LBP/BPI的家长转移保护了鸡眼疟原虫 卵菌感染。 PLoS Pathog 9(12):e1003792。
  • 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: © 2014 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. Baron, O. L. and Pauron, D. (2014). Protein-lipid Interaction Analysis by Surface Plasmon Resonance (SPR). Bio-protocol 4(18): e1237. DOI: 10.21769/BioProtoc.1237.
  2. Baron, O. L., van West, P., Industri, B., Ponchet, M., Dubreuil, G., Gourbal, B., Reichhart, J. M. and Coustau, C. (2013). Parental transfer of the antimicrobial protein LBP/BPI protects Biomphalaria glabrata eggs against oomycete infections. PLoS Pathog 9(12): e1003792.
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。