Laboratory of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic.
J Immunol Methods. 2013 Sep 30;395(1-2):63-70. doi: 10.1016/j.jim.2013.07.001. Epub 2013 Jul 10.
Microtubules formed by αβ-tubulin dimers represent cellular structures that are indispensable for the maintenance of cell morphology and for cell motility generation. Microtubules in intact cells are in highly regulated equilibrium with cellular pools of soluble tubulin dimers. Sensitive, reproducible and rapid assays are necessary to monitor tubulin changes in cytosolic pools after treatment with anti-mitotic drugs, during the cell cycle or activation and differentiation events. Here we describe new assays for α-tubulin quantification. The assays are based on sandwich ELISA, and the signal is amplified with biotinyl-tyramide or immuno-PCR. Matching monoclonal antibody pair recognizes phylogenetically highly conserved epitopes localized outside the C-terminal isotype-defining region. This makes it possible to detect α-tubulin isotypes in different cell types of various species. Biotinyl-tyramide amplification and immuno-PCR amplification enable detection of tubulin at concentrations 2.5ng/ml and 0.086ng/ml, respectively. Immuno-PCR detection shows enhanced sensitivity and wider dynamic range when compared to ELISA with biotinyl-tyramide detection. Our results on taxol-treated and activated bone marrow-derived mast cells demonstrate, that the assays allow sensitive quantification of tubulin in complex biological fluids.
微管由αβ-微管蛋白二聚体组成，是维持细胞形态和产生细胞运动所必需的细胞结构。完整细胞中的微管与细胞内可溶性微管蛋白二聚体池处于高度调节平衡状态。为了监测抗有丝分裂药物处理后、细胞周期中或激活和分化事件期间细胞浆中微管的变化，需要灵敏、可重复和快速的测定法。在这里，我们描述了用于α-微管蛋白定量的新测定法。该测定法基于夹心 ELISA，信号通过生物素化酪胺或免疫 PCR 进行放大。配对的单克隆抗体识别种系高度保守的表位，定位于 C 末端同种型定义区域之外。这使得能够在不同物种的不同细胞类型中检测到α-微管蛋白同型物。生物素化酪胺放大和免疫 PCR 放大可分别检测到浓度为 2.5ng/ml 和 0.086ng/ml 的微管蛋白。与生物素化酪胺检测的 ELISA 相比，免疫 PCR 检测显示出更高的灵敏度和更宽的动态范围。我们在紫杉醇处理和激活的骨髓来源的肥大细胞上的结果表明，该测定法允许在复杂的生物液中敏感地定量微管蛋白。
