Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells.
Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells.
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肌球蛋白在细胞质，裂解沟和人类细胞有丝分裂纺锤体中的荧光抗体定位。
DOI：
10.1083/jcb.71.3.848
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发表时间：
1976-12
影响因子：
7.8
通讯作者：
Pollard, T D
中科院分区：
文献类型：
--
作者：
Fujiwara, K;Pollard, T D
我们利用肌球蛋白特异性抗体偶联荧光染料研究了肌球蛋白分子在人细胞中的分布。用血小板肌球蛋白或肌球蛋白棒免疫家兔。他们生产的抗血清在血小板粗提物的所有成分中只析出肌球蛋白。我们从这些抗血清中分离出免疫球蛋白G （IgG）并与四甲基罗丹明或荧光素偶联。我们用离子交换色谱法从欠共轭和过共轭的IgG中分离出每分子2-5个荧光色的IgG，并用它对丙酮处理的细胞进行染色。以下对照确定了染色模式的特异性：(a)用标记的免疫前IgG染色；(b)用纯化肌球蛋白吸附的标记免疫IgG染色；(c)用标记的免疫IgG与未标记的免疫前血清或免疫血清混合染色；(d)亲和层析纯化的标记抗体染色。在血液涂片中，只有血小板和白细胞的细胞质被染色。在扩散的Enson和HeLa细胞中，应力纤维在紧密间隔的0.5 μ m斑点中染色强烈。在丙酮处理前，细胞质染色均匀，被认为是可运动的。在HeLa细胞分裂过程中，在收缩环附近和有丝分裂纺锤体，特别是染色体和两极之间的区域，有高浓度的肌球蛋白特异性染色。我们没有检测到红细胞、白细胞和培养细胞的细胞核、血小板和培养细胞的表面染色。
We have studied the distribution of myosin molecules in human cells using myosin-specific antibody coupled with fluorescent dyes. Rabbits were immunized with platelet myosin or myosin rod. They produced antisera which precipitated only myosin among all the components in crude platelet extracts. From these antisera we isolated immunoglobulin- G (IgG) and conjugated it with tetramethylrhodamine or fluorescein. We separated IgG with 2-5 fluorochromes per molecule from both under- and over-conjugated IgG by ion exchange chromatography and used it to stain acetone-treated cells. The following controls established the specificity of the staining patterns: (a) staining with labeled preimmune IgG; (b) staining with labeled immune IgG adsorbed with purified myosin; (c) staining with labeled immune IgG mixed with either unlabeled preimmune or immune serum; and (d) staining with labeled antibody purified by affinity chromatography. In blood smears, only the cytoplasm of platelets and leukocytes stained. In spread Enson and HeLa cells, stress fibers stained strongly in closely spaced 0.5 mum spots. The cytoplasm stained uniformly in those cells presumed to be motile before acetone treatment. In dividing HeLa cells there was a high concentration of myosin-specific staining in the vicinity of the contractole ring and in the mitotic spindle, especially the region between the chromosomes and the poles. We detected no staining of erythrocytes, or nuclei of leukocytes and cultured cells, or the surface of platelets and cultured cells.