高速离心-同位素示踪法分析核糖体-mRNA复合物

Analysis method of ribosome-mRNA complex

本实验全面改进了用于蛋白质起始翻译阶段研究的传统介质梯度离心法。以3 2 P (TMV -RNA)为供试mRNA在麦胚离体翻译系统中形成核糖体 mRNA复合物 ,在 10 0 μL蔗糖垫上 (含蔗糖、Hepes、KAC、Mg(AC) 2 和肝素钠 ,pH7.5 ) ,1.5× 10 4r·min-1离心 15 0min即可分离该复合物与游离mRNA。测定沉淀物的cpm值可对其定性定量分析。该方法简便快速、准确安全。

The traditional sucrose gradient centrifugation for protein initiation translation analysis was improved greatly. 1μg 32 P (TMV-RNA) was reacted with 25μL wheat germ extraction, then the mixture was centrifuged for 150min at 1.5×10 4r·min -1 through 100μL sucrose cushions (sucrose 0.5mol·L -1 , Hepes 25mmol·L -1 , KAC 80mmol·L -1 , Mg(AC) 2 1mmol·L -1 , heparin sodium 0.1mmol·L -1 , pH7.5). The sediment included ribosome (TMV RNA) complex and analyzed by liquid scintillator. The method was proved to be sample, quick, sensitive, cheap and safe.