搜索

Proximity Ligation Assay (PLA) to Detect Protein-protein Interactions in Breast Cancer
邻位连接技术(PLA)检测乳腺癌中蛋白质的相互作用   

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

Protein-protein interaction networks provide a global picture of cellular function and biological processes, and the dysfunction of some interactions causes many diseases, including cancer. The in situ proximity ligation assay (PLA) is a powerful technology capable of detecting the interactions among proteins in fixed tissue and cell samples. The interaction between two proteins is detected using the corresponding two primary antibodies raised in different species. Species-specific secondary antibodies (PLA probes), each with a unique short DNA strand attached to it, bind to the primary antibodies. When the PLA probes are in close proximity (<40 nm), the DNA strands can interact through a subsequent addition of two other circle-forming DNA oligonucleotides. Several-hundredfold replication of the DNA circle can occur after the amplification reaction, and a fluorescent signal is generated by labelled complementary oligonucleotide probes. Therefore, each detected signal is visualized as an individual fluorescent dot, which can be quantified and assigned to a specific subcellular location based on microscopy images. This revolutionary technique enables us to study the protein complex formation with high specificity and sensitivity compared to the other traditional methods, such as co-immunoprecipitation (Co-IP).

Materials and Reagents

  1. Antibody pair for detecting protein-protein interaction (two primary antibodies must have been raised in different species, e.g. rabbit-anti-EGFR from Cell Signaling Technology, catalog number: 2232 ; mouse-anti-DNA-PK from BD Pharmingen, catalog number: 556456 )
  2. DuolinkTM in situ reagents
    1. DuolinkTM in situ PLA reagents
      1. Duolink in situ complementary oligonucleotide probe MINUS and PLUS [5x, secondary antibody conjugated with a PLA oligonucleotide, the choice of PLA probes depends on the species of your primary antibodies: e.g. anti-rabbit PLUS (Sigma-Aldrich, catalog number: DUO82029 ); anti-mouse MINUS (Sigma-Aldrich, catalog number: DUO 92004)].
      2. Blocking solution (Sigma-Aldrich, catalog number: DUO82014 ): For blocking of the sample if you have not already optimized your primary antibody with another blocking solution, e.g. 2% BSA in PBS.
      3. Antibody diluent (Sigma-Aldrich, catalog number: DUO82015 ): For dilution of PLA probes and the primary antibodies, alternatively PBS solution with 1% BSA works as well.
    2. Duolink detection reagents
      1. Ligation reagents (5x, contains oligonucleotides that hybridize to the PLA probes and all components needed for ligation except the Ligase) (Sigma-Aldrich, catalog number: DUO82016 )
      2. Ligase (1 unit/μl) (Sigma-Aldrich, catalog number: DUO82029)
      3. Amplification reagents (5x, contains all components needed for Rolling Circle Amplification except the Polymerase. Included are also oligonucleotide probes labelled with a fluorophore that hybridize to the RCA product): Containing far red fluorescently labelled oligonucleotides (Sigma-Aldrich, catalog number: DUO82019 )
      4. Polymerase (10 unit/μl): (Sigma-Aldrich, catalog number: DUO82030 )
        The Duolink detection reagents are available to purchase as a kit from Sigma-Aldrich (e.g. catalog number for Duolink in situ Detection Reagents Far Red DUO92013 ).
    3. Duolink washing buffer A and B (see recipes)
  3. ProLong Gold antifade reagent with DAPI (Life Technologies, catalog number: P-36931), alternatively regular immunofluorescence mounting media and DAPI can be used separately.
  4. Reagents required for fixation and permeabilization of the sample (e.g. 3.7% fresh-made formaldehyde and 0.1% Triton X-100 diluted in PBS)

Equipment

  1. Fluorescence microscope equipped as follows (e.g. Leica TCS SP5 Microsystems)
    1. Excitation/emission filters compatible with fluorophore (ranging from 488-633 nm) and nuclear stain (ultraviolet) excitation/emission
    2. Camera and software for image acquisition
  2. Shaker
  3. Humidity chamber (moist chamber) (Figure 1)
  4. Freeze block for enzymes
  5. 37 °C incubator
  6. Pipettes (covering the range from 1 μl to 1,000 μl)
  7. Glass cover slips compatible with fluorescence microscopy (12 mm diameter and 0.13 to 0.16 mm thickness) (Optics, catalog number: 01 115 20 )
  8. MilliQ® or other equivalently high purity water

Software

  1. Duolink ImageTool software is highly recommended (Demo version for free-download: http://www.olink.com/products/duolink/downloads/duolink-image-tool). Alternatively, ImageJ or LAS AF Lite software from Leica Microsystems can be used for analysis.

Procedure

  1. The primary antibodies need to be optimized through concentration titration to make sure that they work properly before the start of proximity ligation assay (usually ranging from 1:100 to 1:500), and IgG from the same isotype and species was usually used as negative control.
  2. Breast cancer cells (e.g. Hs578T and MDA-MB-468) were transferred on the coverslip in plates/culture dishes and grown to 50%-70% confluency.
  3. The cells were washed with PBS, fixed with 3.7% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, washed, and then blocked using Duolink blocking buffer for 1 h (all these steps are usually performed at room temperature; if you have previously optimized your assay, use the same conditions for Duolink).
  4. The coverslips were transferred into a humidified chamber (Figure 1) and then a small volume of primary antibodies targeting the proteins under investigation were pipetted onto each coverslip (for a 12 mm coverslip, 20-40 µl of antibody added in antibody diluent is more than enough). Antigen-antibody interaction will mostly accomplish in an hour at room temperature, or overnight at 4 °C with gentle agitation (the residual solution can be absorbed using tissues to obtain an equal residual volume on each slide as this will affect reproducibility, However, do not allow the samples to dry before adding the primary antibodies as this will cause background).
  5. The two corresponding PLA probes were mixed and diluted (1:5) in the antibody diluent. Allow the mixture to sit for 20 min at room temperature (e.g. for a 50 μl reaction take 10 μl of PLA probe MINUS stock, 10 μl of PLA probe PLUS stock and 30 μl of the antibody diluent). The samples were washed with PBS+0.05% Tween twice, and then incubated with PLA probes MINUS and PLUS for 1 h at 37 °C.
  6. The samples were washed in 1x wash buffer A for 2 x 5 min under gentle agitation, and the probes were ligated with two other circle-forming DNA oligonucleotides by ligation-ligase solution for 30 min at 37 °C (1:40 dilution, consisting of two oligonucleotides and ligase to hybridize oligonucleotides to the two PLA probes and join to a closed circle if they are in close proximity).
  7. The samples were washed in 1x wash buffer A for 2 x 2 min under gentle agitation and two added oligonucleotides by enzymatic ligation were amplified via rolling circle amplification by the incubation with amplification-polymerase solution (1:80 dilution) over 90 min at 37 °C (consisting of nucleotides and fluorescently labelled oligonucleotides, which is added together with polymerase. The fluorescently labelled oligonucleotides will hybridize to the rolling-circle amplification (RCA) product using the ligated circle as a template, and the signal is easily visible as a distinct fluorescent spot by fluorescence microscope).
  8. The samples were washed in 1x wash buffer B for 2 x 10 min followed by 0.01x wash buffer B for 1 min by diluting 1x buffer B 1:100 in high purity water.
    Note: In steps 6-8 above, the volume of wash buffers A and B depends on the size of coverslip, for a small one (12 mm), we usually wash in a 12-well plate using 1 ml wash buffer.
  9. The samples were dried in hood for approximately 10 min at room temperature in the dark, and mounted onto a slide in a minimal volume of ProLong Gold antifade reagent with DAPI.
  10. Fluorescent signal amplification was used for the assay and interactions were detected using a fluorescence or confocal microscope, using at least a 20x objective. After imaging, the slides can be stored at -20 °C in the dark.


    Figure 1. The chamber for incubation. The 6-well plate was covered with a piece of parafilm to support the coverslip with cells, and another lid was used to prevent sample drying. The chamber is designed to be incubated in a tissue culture incubator at 37 °C for most of the PLA reactions.

Representative data



Figure 2. Duolink proximity ligation assay for protein interactions between epidermal growth factor receptor (EGFR) and DNA-dependent protein kinase (DNA-PK) in breast cancer cell line MDA-MB-468. The cells were treated with DNA-damaging agent etoposide (20 μM) at different time points, and then Duolink assay between EGFR and IGFBP-3 performed as described (cells without treatment as negative controls). Each red spot represents for a single interaction and DNA was stained with DAPI.

Recipes

  1. Duolink in situ wash buffer A
    8.8 g NaCl
    1.2 g Tris base
    0.5 ml Tween 20
    Adjust pH to 7.4 with HCl
    Add high purity dH2O to 1,000 ml (final concentrations 0.01 M Tris, 0.15 M NaCl and 0.05% Tween 20)
    Filter sterilize (0.22 μm)
    Stored at 4 °C, bring the solutions to room temperature before use
  2. Duolink in situ wash buffer B
    5.84 g NaCl
    4.24 g Tris base
    26.0 g Tris-HCl
    Adjust pH to 7.5 using HCl
    Add high purity dH2O to 1,000 ml (final concentrations 0.2 M Tris and 0.1M NaCl)
    Filter the solution through a 0.22 μm filter
    Stored at 4 °C. Bring the solutions to room temperature before use

Acknowledgments

This work was supported by Grant Number DP0984232 to RCB from the Australian Research Council.

References

  1. Soderberg, O., Gullberg, M., Jarvius, M., Ridderstrale, K., Leuchowius, K. J., Jarvius, J., Wester, K., Hydbring, P., Bahram, F., Larsson, L. G. and Landegren, U. (2006). Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3(12): 995-1000.

简介

蛋白质 - 蛋白质相互作用网络提供细胞功能和生物过程的全局图像,并且一些相互作用的功能障碍引起许多疾病,包括癌症。原位接近连接测定(PLA)是一种能够检测固定组织和细胞样品中蛋白质之间相互作用的强大技术。使用在不同物种中产生的相应的两种一抗来检测两种蛋白质之间的相互作用。物种特异性二抗(PLA探针),每个具有连接到其的独特的短DNA链,结合一抗。当PLA探针非常接近(<40nm)时,DNA链可以通过随后添加两个其它形成环的DNA寡核苷酸相互作用。 DNA环的几百倍复制可以在扩增反应之后发生,并且通过标记的互补寡核苷酸探针产生荧光信号。因此,每个检测到的信号被可视化为单个荧光点,其可以基于显微镜图像被量化并分配到特定的亚细胞位置。这种革命性的技术使我们能够与其他传统方法,如共免疫沉淀(Co-IP)相比,以高特异性和灵敏度研究蛋白质复合物形成。

材料和试剂

  1. 用于检测蛋白质 - 蛋白质相互作用的抗体对(两种一抗必须在不同物种中产生,例如来自Cell Signaling Technology的兔抗EGFR,目录号:2232;小鼠抗DNA-PK 来自BD Pharmingen,目录号:556456)
  2. Duolink TM 原位试剂
    1. Duolink TM 原位 PLA试剂
      1. Duolink原位互补 寡核苷酸探针MINUS和PLUS [5x,二抗偶联 与PLA寡核苷酸,PLA探针的选择取决于 您的主要抗体种类:例如抗兔PLUS (Sigma-Aldrich,目录号:DUO82029); 抗鼠MINUS (Sigma-Aldrich,目录号:DUO 92004)]
      2. 封闭溶液 (Sigma-Aldrich,目录号:DUO82014):用于阻断样品if   你还没有用另一个优化你的一级抗体 封闭溶液,例如PBS中的2%BSA。
      3. 抗体稀释液 (Sigma-Aldrich,目录号:DUO82015):用于稀释PLA探针 和第一抗体,或者具有1%BSA的PBS溶液   以及。
    2. Duolink检测试剂
      1. 连接试剂 (5x,包含与PLA探针杂交的寡核苷酸和所有 除了连接酶之外的连接所需的组分)(Sigma-Aldrich, 目录号:DUO82016)
      2. 连接酶(1单位/μl)(Sigma-Aldrich,目录号:DUO82029)
      3. 扩增试剂(5x,包含所需的所有组分 除聚合酶外的滚环扩增。 包括也 用与之杂交的荧光团标记的寡核苷酸探针   RCA产品):含有远红色荧光标记 寡核苷酸(Sigma-Aldrich,目录号:DUO82019)
      4. 聚合酶(10单位/μl):( Sigma-Aldrich,目录号:DUO82030)
        Duolink检测试剂可作为试剂盒购买 Sigma-Aldrich(例如 Duolink 原位检测的目录号 试剂远红DUO92013)。
    3. Duolink洗涤缓冲液A和B(见配方)
  3. 用DAPI(Life Technologies,目录号:P-36931)的ProLong Gold抗衰减试剂,或者可以单独使用常规免疫荧光封固剂和DAPI。
  4. 试剂固定和透化所需的试剂(例如3.7%新鲜制备的甲醛和稀释在PBS中的0.1%Triton X-100)

Duolink检测试剂可作为试剂盒购买 Sigma-Aldrich(例如 Duolink 原位检测的目录号 试剂远红DUO92013)。

  • Duolink洗涤缓冲液A和B(见配方)
  • 用DAPI(Life Technologies,目录号:P-36931)的ProLong Gold抗衰减试剂,或者可以单独使用常规免疫荧光封固剂和DAPI。
  • 试剂固定和透化所需的试剂(例如3.7%新鲜制备的甲醛和稀释在PBS中的0.1%Triton X-100)
  • ... Duolink检测试剂可作为试剂盒购买 Sigma-Aldrich(例如 Duolink 原位检测的目录号 试剂远红DUO92013)。

  • Duolink洗涤缓冲液A和B(见配方)
  • 用DAPI(Life Technologies,目录号:P-36931)的ProLong Gold抗衰减试剂,或者可以单独使用常规免疫荧光封固剂和DAPI。
  • 试剂固定和透化所需的试剂(例如3.7%新鲜制备的甲醛和稀释在PBS中的0.1%Triton X-100)
  • ...... Duolink检测试剂可作为试剂盒购买 Sigma-Aldrich(例如 Duolink 原位检测的目录号 试剂远红DUO92013)。

  • Duolink洗涤缓冲液A和B(见配方)
  • 用DAPI(Life Technologies,目录号:P-36931)的ProLong Gold抗衰减试剂,或者可以单独使用常规免疫荧光封固剂和DAPI。
  • 试剂固定和透化所需的试剂(例如3.7%新鲜制备的甲醛和稀释在PBS中的0.1%Triton X-100)
  • .........

  • 用PBS洗涤细胞,用3.7%甲醛固定15分钟,用0.1%Triton X-100透化10分钟,洗涤,然后用Duolink封闭缓冲液封闭1小时(所有这些步骤通常在室温下进行;如果您已经优化了您的测定,使用相同的条件Duolink)。
  • 将盖玻片转移到加湿室中(图1),然后将小体积靶向研究中的蛋白质的一级抗体移液到每个盖玻片上(对于12mm盖玻片,加入抗体稀释液中的20-40μl抗体大于足够)。抗原 - 抗体相互作用将主要在室温下在1小时内完成,或在4℃下在轻微搅拌下过夜(残余溶液可以使用组织吸收以在每个载玻片上获得相等的残余体积,因为这将影响重现性。然而,在添加一抗之前不允许样品干燥,因为这将导致背景)。
  • 将两种相应的PLA探针混合并在抗体稀释液中稀释(1:5)。使混合物在室温下静置20分钟(例如,对于50μl反应,取10μlPLA探针MINUS原液,10μlPLA探针PLUS原液和30μl抗体稀释液)。样品用PBS + 0.05%Tween洗涤两次,然后与PLA探针MINUS和PLUS在37℃孵育1小时。
  • 在温和搅拌下,将样品在1×洗涤缓冲液A中洗涤2×5分钟,并且通过连接 - 连接酶溶液将探针与另外两个形成环的DNA寡核苷酸在37℃下连接30分钟(1:40稀释,包括的两个寡核苷酸和连接酶以将寡核苷酸杂交到两个PLA探针,并且如果它们非常接近则连接到闭合圆)。
  • 将样品在1×洗涤缓冲液A中在轻轻搅拌下洗涤2×2分钟,通过酶连接的两个添加的寡核苷酸通过滚环扩增通过与扩增 - 聚合酶溶液(1:80稀释)在37℃下90分钟(由核苷酸和荧光标记的寡核苷酸组成,其与聚合酶一起加入)。荧光标记的寡核苷酸将与连接的圆作为模板,并且信号作为荧光显微镜的不同荧光点容易可见)。
  • 样品在1x洗涤缓冲液B中洗涤2×10分钟,然后通过在高纯度水中稀释1×缓冲液B 1:100洗涤0.01x洗涤缓冲液B 1分钟。
    注意:在上述步骤6-8中,洗涤缓冲液A和B的体积取决于盖玻片的大小,对于小的(12mm),我们通常在12孔板中使用1ml洗涤缓冲液。
  • 将样品在室温下在黑暗中在通风橱中干燥约10分钟,并且在具有DAPI的最小体积的ProLong Gold抗衰退试剂中安装到载玻片上。
  • 荧光信号扩增用于测定,并且使用荧光或共焦显微镜,使用至少20x物镜检测相互作用。成像后,载玻片可在-20℃下避光保存

    图1.孵育室。用一块石蜡膜覆盖6孔板以用细胞支撑盖玻片,并使用另一个盖子来防止样品干燥。室被设计为在组织培养箱中在37℃下孵育大部分PLA反应。
  • 代表数据



    图2.乳腺癌细胞系MDA-MB-468中表皮生长因子受体(EGFR)和DNA依赖性蛋白激酶(DNA-PK)之间的蛋白质相互作用的Duolink邻近连接测定。 在不同时间点用DNA损伤剂依托泊苷(20μM)处理,然后如所述进行EGFR和IGFBP-3之间的Duolink测定(作为阴性对照的细胞)。 每个红色斑点代表单一相互作用,DNA用DAPI染色。

    食谱

    1. Duolink 原位清洗缓冲液A
      8.8克NaCl 1.2克Tris碱
      0.5ml吐温20 用HCl
      调节pH至7.4 将高纯度dH 2 O加至1,000ml(终浓度0.01M Tris,0.15M NaCl和0.05%Tween 20)中
      过滤灭菌(0.22μm)
      储存于4°C,使用前将溶液置于室温下
    2. Duolink 原位洗涤缓冲液B
      5.84g NaCl
      4.24g Tris碱
      26.0g Tris-HCl
      使用HCl
      调节pH至7.5 将高纯度dH 2 O加至1,000ml(终浓度0.2M Tris和0.1M NaCl)中
      通过0.22μm过滤器过滤溶液
      储存于4°C。 使用前将溶液置于室温下

    致谢

    这项工作是由澳大利亚研究委员会的RCB拨款号DP0984232支持的。

    参考文献

    1. Soderberg,O.,Gullberg,M.,Jarvius,M.,Ridderstrale,K.,Leuchowius,KJ,Jarvius,J.,Wester,K.,Hydbring,P.,Bahram,F.,Larsson,LGand Landegren, U.(2006)。 通过邻近连接直接观察单个内源性蛋白复合物 /a> Nat Methods 3(12):995-1000。
    • 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: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
    引用:Lin, M. Z., Martin, J. L. and Baxter, R. C. (2015). Proximity Ligation Assay (PLA) to Detect Protein-protein Interactions in Breast Cancer . Bio-protocol 5(10): e1479. DOI: 10.21769/BioProtoc.1479.
    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。