Nesbitt S A, Horton M A
Imperial Cancer Research Fund, St. Bartholomew's Hospital, London, United Kingdom.
Anal Biochem. 1992 Nov 1;206(2):267-72. doi: 10.1016/0003-2697(92)90365-e.
Here we demonstrate a nonradioactive immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique which replaces the standard practice of isotopic protein labeling by iodination or metabolic tagging in the analysis of membrane proteins. The technique has proved extremely valuable in the biochemical analysis of small quantities of frozen, pathological tissue. Membranes were prepared from Dx3 (a human melanoma cell line), C6 (a rat glial cell line), and osteoclastoma (a human giant cell tumor of bone). The membranes were labeled with biotin and immunoprecipitated with a variety of antibodies to the vitronectin receptor (VNR). The VNR proteins were resolved by SDS-PAGE and immunoblotted onto nitrocellulose paper. The biotinylated protein was visualized using streptavidin horseradish peroxidase and enhanced chemiluminescence (ECL). Film exposures ranged from 15 min to 16 h. Good visualization of the VNR, yielding the typical heterodimeric receptor of 90 and 150 kDa, was given. Signals generated were high and background noise low with a 30-min film exposure. An overnight exposure increased the detection of weaker bands. In conclusion, biotinylation of membrane proteins proved a satisfactory label for immunoprecipitation and SDS-PAGE analysis. The ECL development stage was extremely flexible with visualization of strong and weak signals. The method has several advantages over a conventional radioactive immunoprecipitation in that it is relatively inexpensive, simple, quick and nonhazardous.
在此，我们展示了一种非放射性免疫沉淀和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）技术，该技术在膜蛋白分析中取代了通过碘化或代谢标记进行同位素蛋白标记的标准做法。该技术已被证明在少量冷冻病理组织的生化分析中极具价值。膜取自Dx3（一种人类黑色素瘤细胞系）、C6（一种大鼠神经胶质细胞系）和成骨细胞瘤（一种人类骨巨细胞瘤）。膜用生物素标记，并用多种抗玻连蛋白受体（VNR）抗体进行免疫沉淀。VNR蛋白通过SDS-PAGE分离，并免疫印迹到硝酸纤维素纸上。使用链霉亲和素辣根过氧化物酶和增强化学发光（ECL）对生物素化蛋白进行可视化。胶片曝光时间从15分钟到16小时不等。获得了VNR的良好可视化效果，呈现出典型的90 kDa和150 kDa异二聚体受体。30分钟的胶片曝光产生的信号强，背景噪声低。过夜曝光增加了较弱条带的检测。总之，膜蛋白的生物素化被证明是免疫沉淀和SDS-PAGE分析的一种令人满意的标记。ECL显影阶段极具灵活性，可对强弱信号进行可视化。该方法相对于传统放射性免疫沉淀具有多个优点，即相对便宜、简单、快速且无危害。
