Amartely Hadar, Some Daniel, Tsadok Ayala, Lebendiker Mario
Wolfson Centre for Applied Structural Biology, The Alexander Silberman Institute of Life Science, The Hebrew University of Jerusalem.
Wyatt Technology Corporation.
J Vis Exp. 2019 Apr 5(146). doi: 10.3791/59408.
Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique. Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.
多角度光散射离子交换色谱法（IEX-MALS）是一种用于蛋白质分离和表征的强大方法。高特异性分离技术IEX与通过MALS实现的精确摩尔质量分析相结合，能够对异质蛋白质样品进行表征，包括寡聚体形式或蛋白质群体的混合物，即使它们的摩尔质量非常相似。因此，IEX-MALS提供了额外的蛋白质表征水平，并且是对标准多角度光散射尺寸排阻色谱法（SEC-MALS）技术的补充。在这里，我们描述了一个基本的IEX-MALS实验方案，并在牛血清白蛋白（BSA）上演示了该方法。IEX将BSA分离成其寡聚体形式，从而允许通过MALS对每种单独形式进行摩尔质量分析。还介绍了IEX-MALS实验的优化，并在BSA上进行了演示，实现了BSA单体与较大寡聚体之间的出色分离。IEX-MALS是一种用于蛋白质质量评估的有价值技术，因为它既提供了对样品中存在的多种蛋白质种类的精细分离，又能测定其摩尔质量。
