Ball R L, Carney D H, Albrecht T, Asai D J, Thompson W C
J Cell Biol. 1986 Sep;103(3):1033-41. doi: 10.1083/jcb.103.3.1033.
To detect changes in the extent of tubulin polymerization in cultured cells, we have developed a radioactive antibody binding assay that can be used to quantitate total cytoskeletal tubulin or specific antigenic subsets of polymerized tubulin. Fibroblastic cells, grown to confluence in multiwell plates, were permeabilized and extracted with 0.5% Triton X-100 in a microtubule-stabilizing buffer. These extracted cytoskeletons were then fixed and incubated with translationally radiolabeled monoclonal antitubulin antibody (Ab 1-1.1), an IgM antibody specific for the beta subunit of tubulin. Specific binding of Ab 1-1.1 to the cytoskeletons was saturable and of a single apparent affinity. All specific binding was blocked by preincubation of the radiolabeled antibody with excess purified brain tubulin. Specific Ab 1-1.1 binding appeared to represent binding to cytoskeletal tubulin inasmuch as: pretreatment of cells with colchicine decreased Ab 1-1.1 binding in a dose-dependent manner which correlated with the amount of polymerized tubulin visualized in parallel cultures by indirect immunofluorescence, taxol pretreatment alone caused an increase in Ab 1-1.1 binding and prevented in a dose-dependent manner the colchicine-induced decrease in antibody binding, in cells pretreated with colcemid and returned to fresh medium, Ab 1-1.1 binding decreased and recovered in parallel with the depolymerization and regrowth of microtubules in these cells, and comparison of maximal antibody binding per cell between primary mouse embryo, 3T3, and human foreskin fibroblasts correlated with immunofluorescence visualization of microtubules in these cells. Thus, this assay can be used to measure relative changes in the level of polymerized cytoskeletal tubulin. Moreover, by Scatchard-type analysis of the binding data it is possible to estimate the total number of antibody binding sites per cell. Therefore, depending on the stoichiometry of antibody binding, this type of assay may be used for quantitating total cytoskeletal tubulin, specific antigenic subsets of cytoskeletal tubulin, or other cytoskeletal proteins.
为了检测培养细胞中微管蛋白聚合程度的变化，我们开发了一种放射性抗体结合测定法，该方法可用于定量总细胞骨架微管蛋白或聚合微管蛋白的特定抗原亚群。在多孔板中生长至汇合的成纤维细胞，在微管稳定缓冲液中用0.5% Triton X-100进行通透处理并提取。然后将这些提取的细胞骨架固定，并用经翻译放射性标记的抗微管蛋白单克隆抗体（Ab 1-1.1）孵育，Ab 1-1.1是一种对微管蛋白β亚基具有特异性的IgM抗体。Ab 1-1.1与细胞骨架的特异性结合是可饱和的，且具有单一的表观亲和力。所有特异性结合都可通过将放射性标记抗体与过量纯化的脑微管蛋白预孵育来阻断。特异性Ab 1-1.1结合似乎代表了与细胞骨架微管蛋白的结合，因为：用秋水仙碱预处理细胞会以剂量依赖的方式降低Ab 1-1.1结合，这与通过间接免疫荧光在平行培养物中观察到的聚合微管蛋白量相关；单独用紫杉醇预处理会导致Ab 1-1.1结合增加，并以剂量依赖的方式阻止秋水仙碱诱导的抗体结合减少；在用秋水仙酰胺预处理并返回新鲜培养基的细胞中，Ab 1-1.1结合减少并与这些细胞中微管的解聚和重新生长平行恢复；并且对原代小鼠胚胎、3T3和人包皮成纤维细胞中每个细胞的最大抗体结合进行比较，与这些细胞中微管的免疫荧光观察结果相关。因此，该测定法可用于测量聚合细胞骨架微管蛋白水平的相对变化。此外，通过对结合数据进行Scatchard型分析，可以估计每个细胞的抗体结合位点总数。因此，根据抗体结合的化学计量，这种类型的测定法可用于定量总细胞骨架微管蛋白、细胞骨架微管蛋白的特定抗原亚群或其他细胞骨架蛋白。
