Nowak T S, Carty E R, Lust W D, Passonneau J V
Anal Biochem. 1984 Feb;136(2):285-92. doi: 10.1016/0003-2697(84)90218-5.
A quantitative in vitro amino acid incorporation assay is described which can be used to assess the status of in vivo protein synthesis. The preparation and incubation conditions employed result in constant precursor specific activity and limit amino acid incorporation to completion of nascent peptide chains. Results obtained with this method correlate well with measurements of polyribosome profiles using sucrose gradient centrifugation. The assay is easily applied to a large number of samples, and requires only a fraction of the time and tissue necessary for conventional measures of polysome aggregation. The method has been found suitable for studies of protein synthesis in mouse brain and liver, and in gerbil brain, but not in mouse kidney. Products of in vitro protein synthesis can be separated by standard electrophoretic techniques, allowing a characterization of proteins whose mRNAs are actively translated in vivo.
本文描述了一种定量体外氨基酸掺入测定法，可用于评估体内蛋白质合成的状态。所采用的制备和孵育条件可使前体比活性保持恒定，并将氨基酸掺入限制在新生肽链完成时。用该方法获得的结果与使用蔗糖梯度离心法测量多核糖体图谱的结果相关性良好。该测定法易于应用于大量样品，并且只需要传统多核糖体聚集测量所需时间和组织的一小部分。已发现该方法适用于小鼠脑和肝脏以及沙鼠脑中蛋白质合成的研究，但不适用于小鼠肾脏。体外蛋白质合成产物可通过标准电泳技术分离，从而能够对其mRNA在体内被积极翻译的蛋白质进行表征。
