超速离心-沉降速率法测定蛋白质分子量的实验条件

分子量是蛋白质的重要特征参数,准确测定蛋白质分子量对研究蛋白质结构与功能具有重要意义。该文以牛血清蛋白(BSA)和人血清免疫球蛋白(IgG)为研究对象,采用分析超速离心-沉降速率法(AUC-SV)测定2种蛋白的分子量,并系统研究了样品初始浓度、离心转速、离心时间、离心温度、样品上样体积和缓冲液体系等实验条件对测定结果的影响。结果表明:离心转速为20000~40000 r/min时,影响不明显,样品初始浓度、离心时间、离心温度、缓冲液体系的适宜范围较宽,样品上样体积对测定结果的影响显著。在优化的实验条件下,测得未知蛋白的分子量为39.9 kDa,与质谱法测定结果(40.0 kDa)一致。该方法具有适宜条件范围宽、非破坏性、无需标准品和普适性高等优点,适用于未知蛋白分子量的测定。此外,相关研究结论还可供AUC-SV初学者学习借鉴。

Experimental conditions for determining protein molecular weight by analytical ultracentrifugation sedimentation velocity method

Protein molecular weight is an important characteristic parameter.Accurate determination of protein molecular weight is of great significance to study the structure and function of protein.This work takes albumin bovine serum(BSA)and human serum immunoglobulin G(IgG)as research objects and determines the molecular weights of both by sedimentation velocity of analytical ultracentrifugation(AUC・SV).It aims to investigate the impact of experimental conditions,such as sample initial concentration,centrifugal speed,centrifugal time,centrifugal temperature,sample loading volume and buffer component on determination results.The results show that the effect of centrifugal speed in the range of 20000-40000 r/min on determination results is not obvious,the suitable conditions ranges of sample concentration,centrifugal time,centrifugal temperature and buffer comp on ent are wide,and the sample loading volume has a significant impact.Under the optimized experiment conditions,the molecular weight of unknown protein is 39.9 kDa which is determined by this method.That is consistent with the result(40.0 kDa)determined by mass spectrometry.This method is applicable to the determination molecular weight of unknown proteins,which has wide ranges of suitable conditions,non-destructive,no need for standards and high universality.The research conclusions can also be used as a reference for the beginners to learn AUC-SV.