Folta-Stogniew Ewa
W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven, CT, USA.
Methods Mol Biol. 2006;328:97-112. doi: 10.1385/1-59745-026-X:97.
Size-exclusion chromatography (SEC), coupled with "on-line" static laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection, provides a universal approach for determination of the molar mass and oligomeric state in solution of native proteins as well as glycosylated proteins or membrane proteins solubilized in non-ionic detergents. Such glycosylated proteins or protein-detergent complexes show anomalous behavior on SEC, thus presenting a challenge to determination of molar mass and oligomeric state in solution. In the SEC-UV/LS/RI approach, SEC serves solely as a fractionation step, while the responses from the three detectors are utilized to calculate the molar mass for the polypeptide portion of the native or modified protein. The amount of sugar, lipid, or detergent bound to the polypeptide chain can also be estimated from the SEC-UV/LS/RI analysis.
尺寸排阻色谱法（SEC）与“在线”静态激光光散射（LS）、折射率（RI）和紫外（UV）检测相结合，为测定天然蛋白质以及糖基化蛋白质或溶解于非离子洗涤剂中的膜蛋白在溶液中的摩尔质量和寡聚状态提供了一种通用方法。此类糖基化蛋白质或蛋白质 - 洗涤剂复合物在SEC上表现出异常行为，因此对溶液中摩尔质量和寡聚状态的测定构成挑战。在SEC - UV/LS/RI方法中，SEC仅作为一个分离步骤，而来自三个检测器的响应则用于计算天然或修饰蛋白质多肽部分的摩尔质量。与多肽链结合的糖、脂质或洗涤剂的量也可以通过SEC - UV/LS/RI分析来估算。
