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分子相互作用研究:荧光相关光谱技术在微升细胞裂解液中一步分析蛋白质复合物

分子相互作用研究:荧光相关光谱技术在微升细胞裂解液中一步分析蛋白质复合物

  • 分类:应用案例
  • 发布时间:2023-01-06 14:21
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【概要描述】德国图宾根大学分子生物学系细胞生物学跨文化研究所Roland Brock团队应用荧光相关光谱技术(FCS & FCCS)和免疫荧光标记法在微量细胞裂解液中定量分析内源性蛋白分子的相互作用。传统研究内源性生物分子相互作用的实验技术(比如免疫共沉淀;Co-IP)的实验过程繁琐并容易忽略弱分子相互作用。针对这一科研关键点和难点,作者首先在鼠源3a9细胞中表达了融合蛋白ZAP-70-YFP,之后使用钒酸盐

分子相互作用研究:荧光相关光谱技术在微升细胞裂解液中一步分析蛋白质复合物

【概要描述】德国图宾根大学分子生物学系细胞生物学跨文化研究所Roland Brock团队应用荧光相关光谱技术(FCS & FCCS)和免疫荧光标记法在微量细胞裂解液中定量分析内源性蛋白分子的相互作用。传统研究内源性生物分子相互作用的实验技术(比如免疫共沉淀;Co-IP)的实验过程繁琐并容易忽略弱分子相互作用。针对这一科研关键点和难点,作者首先在鼠源3a9细胞中表达了融合蛋白ZAP-70-YFP,之后使用钒酸盐

  • 分类:应用案例
  • 发布时间:2023-01-06 14:21
  • 访问量:
详情

德国图宾根大学分子生物学系细胞生物学跨文化研究所Roland Brock团队应用荧光相关光谱技术(FCS & FCCS)和免疫荧光标记法在微量细胞裂解液中定量分析内源性蛋白分子的相互作用。传统研究内源性生物分子相互作用的实验技术(比如免疫共沉淀;Co-IP)的实验过程繁琐并容易忽略弱分子相互作用。针对这一科研关键点和难点,作者首先在鼠源3a9细胞中表达了融合蛋白ZAP-70-YFP,之后使用钒酸盐(PV)处理细胞,应用FCS/FCCS技术验证了融合蛋白ZAP-70-YFP能与受体表面的膜蛋白CD3ε结合。为排除外源性表达蛋白可能导致的假阳性结果,作者应用特异性抗体对内源性蛋白进行荧光标记,FCS/FCCS实验结果显示酪氨酸激酶ZAP-70在PV刺激下可与CD3ε发生相互作用。为进一步研究PV刺激细胞后内源性蛋白的信号传导机制,作者使用FCCS技术研究了未转染的鼠源3a9和人源Jurkat细胞;结果显示:在3a9细胞中,检测到信号蛋白GRB2和PLCγ1与骨架蛋白LAT存在相互作用(+PV);在Jurkat细胞中,GRB2-LAT未检测到互作,但LAT-PLCγ1、SLP-76-PLCγ1存在相互作用(+PV)。最后,作者将以上新技术示范应用于细胞裂解液样品的多肽药物筛选。本文展示了一种仅需在微量细胞裂解液中加入一抗和荧光标记二抗,然后应用荧光相关光谱技术快速、定量分析分子互作的实验新技术。与传统技术(例如CO-IP)相比,FCS/FCCS方法具有样品量少、操作简单、无需样品纯化等优点,对基础生物学研究和药物筛选具重要意义。


Abstract

  We present 'mix and measure' procedures for the analysis of protein complexes in microliters of crude human and mouse cell lysates using fluorescence correlation and crosscorrelation spectroscopy. We labeled interacting endogenous proteins by indirect immunofluorescence with all primary and secondary reagents added in one step. Especially for the screening of compounds interfering with interactions that depend on signaling-induced posttranslational modifications, the approach represents a major advance over existing protocols.


Figure 1 Schematic of protein complex analysis by FCCS and FCS using indirect immunolabeling.(a) FCS derives information from temporal fluctuations of fluorescence caused by the diffusion of fluorescent particles through a confocal detection volume11. Attachment of a mass tag to a fluorophore-labeled particle slows down the diffusion of the particle, resulting in a right shift of the autocorrelation function G(t) toward longer autocorrelation (ac) times12. (b) FCCS detects interactions based on the joint motion of particles tagged with spectrally distinct fluorophores. The higher the fraction of doubly labeled particles, the higher the amplitude Gcc(t= 0) of the crosscorrelation function Gcc(t).(c) Detection of complexes of endogenous proteins, exemplified for the pervanadate-induced (+PV) interaction of ZAP-70 with CD3Ɛ. Two sets of primary antibodies and matching secondary reagents are added to the lysate. One secondary reagent introduces a fluorophore, the other one (I) a second fluorophore or (II) a mass tag. The interaction is detected (I) by the crosscorrelation (cc) or (II) by the right shift of the autocorrelation function.

Figure 2 Detection of stimulation-dependent protein complexes and testing of inhibitors of complex formation. (a)Interaction of ZAP-70 and CD3Ɛ detected by FCCS (lysate: 106 3a9 cells/20 μL). Reagents: mouse anti-ZAP-70 (Z), biotinylated hamster anti-CD3Ɛ (Ɛ),Alexa488-labeled anti-mouse, streptavidin-Cy5. (-), primary antibodies omitted. (b) Mass tag-FCS detection of the ZAP-70-CD3Ɛ interaction (lysate: 2×105 3a9 cells/20 μL). Primary antibodies as in a, nonspecific mouse antibody (iso); secondary reagents: anti-mouse Fab-Alexa647, streptavidin-nanobeads. (c) Further stimulation-dependent interactions of endogenous proteins detected by FCCS (lysates: Jurkat or 3a9, 106 cells/20 μL). Reagents: pairs of primary antibodies, one each from mouse and rabbit, species specific secondary antibodies labeled with Alexa488 or Alexa633. Control samples contained lysis buffer instead of cell lysate. Data from three independent experiments (LAT-GRB2 in Jurkat, from two independent experiments); mean values and error propagations of s.d. of the single experiments. (d) Titration of ZAP-70-YFP-CD3Ɛ complexes with pITAM, added to lysate (2×105 cells/20 μL) of PV-treated cells. Dotted lines, mean crosscorrelation amplitude in lysate of pervanadate-treated cells without peptide (top line), lysate of resting cells (bottom line). (e) Crosscorrelation amplitudes for the titration of the LAT-PLCγ1 interaction in lysates of pervanadate-treated Jurkat cells (106 cells/20μL) with peptides LAT-pY132 and LAT-pY191. Antibodies: mouse anti-LAT, rabbit anti PLCγ1, anti-mouse-Alexa488, anti-rabbit-Alexa633. LAT-Y191: unphosphorylated control peptide. Dotted lines as in d. (f) Schematic of the signaling complex of LAT, SLP-76, PLCγ1 and GADS and the binding sites on LAT mimicked by LAT-pY132 and LAT-pY191 (solid lines). LAT-pY191 might also interfere at the PLCγ1-LAT binding site (dotted line). -PV and +PV, lysate from resting and pervanadate-treated cells, respectively. Average curves (a,b,d) or mean values and standard deviations (d,e) of ten repeated measurements in one representative experiment.


原文链接:https://pubmed.ncbi.nlm.nih.gov/16278653/

 


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