Freeman J W, Hazlewood J E, Auerbach P, Busch H
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030.
Cancer Res. 1988 Sep 15;48(18):5246-50.
Our laboratory has reported (J. W. Freeman et al., Cancer Res., 48:1244-1251, 1988) a proliferation-associated Mr 120,000 nucleolar antigen (p120), that was found in human tumors but was not detectable in most normal resting tissues and in benign tumors. To study further the function and the localization of this protein, we have investigated various methods of microinjecting p120 monoclonal antibodies into cells. For comparison, we have used a monoclonal antibody to protein C23, a nucleolar protein found in high levels in most cells. To determine optimal conditions for loading of antibodies to nucleolar antigens into mechanically disrupted HeLa cells, we studied the effects of ionic strengths of loading buffers, various antibody concentrations, and optimal time for loading and antibody localization. With ascites fluids in isotonic buffer containing antibodies to nucleolar proteins p120 and C23, a maximum number of cells, 86 and 84%, respectively, were loaded following a 20-min incubation. Hypotonic buffers decreased the percentage of cells loaded (22%); hypertonic buffers reduced cell viability. The optimal concentration of purified antibody yielding a maximum number of loaded cells (81%) was 2.5 mg/ml. Higher concentrations of antibody resulted in residual cytoplasmic staining without increasing the percentage of loaded cells. With antibody concentrations less than 2.5 mg/ml, a linear decrease was noted in the percentage of cells loaded with a decrease in intensity of fluorescence. Following antibody loading, nucleolar fluorescence was observed by 12 h and the intensity increased at 24 h. Localization of the p120 antibody was followed through mitosis where it was perichromosomal and equally divided between the chromosomes at metaphase. A decrease of nucleolar immunofluorescence intensity and percentage of cells labeled were observed in successive cell generations.
我们实验室已报道（J. W. 弗里曼等人，《癌症研究》，48:1244 - 1251，1988）一种与增殖相关的分子量为120,000的核仁抗原（p120），它在人类肿瘤中被发现，但在大多数正常静止组织和良性肿瘤中无法检测到。为了进一步研究这种蛋白质的功能和定位，我们研究了将p120单克隆抗体显微注射到细胞中的各种方法。作为比较，我们使用了针对蛋白C23的单克隆抗体，C23是一种在大多数细胞中含量很高的核仁蛋白。为了确定将核仁抗原抗体加载到机械破碎的HeLa细胞中的最佳条件，我们研究了加载缓冲液的离子强度、各种抗体浓度以及加载和抗体定位的最佳时间的影响。使用含有核仁蛋白p120和C23抗体的等渗缓冲液中的腹水，孵育20分钟后，分别有最多数量的细胞（86%和84%）被加载。低渗缓冲液降低了加载细胞的百分比（22%）；高渗缓冲液降低了细胞活力。产生最大加载细胞数量（81%）的纯化抗体的最佳浓度为2.5毫克/毫升。更高浓度的抗体导致残留的细胞质染色，而没有增加加载细胞的百分比。当抗体浓度低于2.5毫克/毫升时，随着荧光强度的降低，加载细胞的百分比呈线性下降。抗体加载后12小时观察到核仁荧光，24小时时强度增加。p120抗体的定位在有丝分裂过程中被追踪，在中期它位于染色体周围并在染色体之间平均分配。在连续的细胞世代中观察到核仁免疫荧光强度和标记细胞百分比的下降。
