Hoffman W L, Ilan J
Biochim Biophys Acta. 1977 Feb 3;474(3):411-24. doi: 10.1016/0005-2787(77)90270-2.
Ribosomal proteins were analyzed by means of two-dimensional gel electrophoresis. To insure that the analysis included only that fraction of the ribosome actively participating in protein synthesis, only polysomal-bound ribosomes were used. This differs from previously reported analyses of liver ribosomal proteins. The ribosomal proteins were prepared from ribosomes of polysomal origin from membrane-bound and free polysomes. Membrane-bound and free liver polysomes were isolated from unfasted mice. The polysomes were purified on hydroxyapatite under conditions known to result in polysomes and ribosomes that are active in both endogenous and synthetic mRNA translation. Moreover, this is the first time that liver ribosomal protein was obtained and analyzed from animals that have not been starved prior to sacrifice. The puromycin-released ribosomes were dissociated into subunits and ribosomal proteins were analyzed by means of two-dimensional polyacrylamide gel electrophoresis. When 100-200 mug samples of the ribosomal subunit proteins were analyzed by two-dimensional electrophoresis, approximately 32 major proteins were detected for the 60 S ribosomal subunit and 25 major proteins for the 40 S ribosomal subunit. A total of 13 "fractional" ribosomal proteins was also detected in the ribosomal subunit profiles. No differences in number or mobility of the ribosomal proteins were found between the membrane-bound and free ribosome populations. We describe a system in which all ribosomal proteins are completely solubilized and quantitatively move from the first to the second dimension gel. Thus the total sample is separated and fractionated. This procedure elimates artifacts due to incomplete solubilization of ribosomal proteins, which is common for the transfer from the first- to second-dimension gel. Therefore, a more detailed and accurate analysis is achieved.
通过二维凝胶电泳对核糖体蛋白进行分析。为确保分析仅包括核糖体中积极参与蛋白质合成的那部分，仅使用与多核糖体结合的核糖体。这与先前报道的肝脏核糖体蛋白分析不同。核糖体蛋白是从膜结合多核糖体和游离多核糖体的多核糖体来源的核糖体中制备的。从未禁食的小鼠中分离出膜结合和游离的肝脏多核糖体。在已知能产生对内源和合成mRNA翻译均有活性的多核糖体和核糖体的条件下，将多核糖体在羟磷灰石上纯化。此外，这是首次从处死前未饥饿的动物中获得并分析肝脏核糖体蛋白。将嘌呤霉素释放的核糖体解离成亚基，并通过二维聚丙烯酰胺凝胶电泳分析核糖体蛋白。当通过二维电泳分析100 - 200微克核糖体亚基蛋白样品时，在60S核糖体亚基中检测到约32种主要蛋白，在40S核糖体亚基中检测到25种主要蛋白。在核糖体亚基图谱中还总共检测到13种“部分”核糖体蛋白。在膜结合核糖体群体和游离核糖体群体之间，未发现核糖体蛋白的数量或迁移率有差异。我们描述了一种系统，其中所有核糖体蛋白都能完全溶解，并定量地从第一维凝胶转移到第二维凝胶。这样整个样品就被分离和分级了。该程序消除了由于核糖体蛋白溶解不完全而产生的假象，这种假象在从第一维凝胶转移到第二维凝胶时很常见。因此，实现了更详细和准确的分析。
