what is difference between size exclusion chromatography, fluroscence size exclusion chromatography (FSEC) and size exclusion chromatography-multi angle light scattering (SEC-MALS)? I want to study stochiecometry and oligomeric state about my protein so which technique is useful?
The stoichiometry of a protein complex can be calculated from an accurate measurement of the complex’s molecular weight. SEC-MALS provides a powerful means for determining the molecular weights of proteins and protein complexes. In contrast to conventional size-exclusion chromatography and analytical centrifugation, measurements do not rely on the use of molecular weight standards and are not affected by the shape of the proteins. Please follow the link for complete info:
https://link.springer.com/protocol/10.1385/1-59259-762-9:113/fulltext.html
Size exclusion chromatography, also called gel filtration chromatography, is a method for separating proteins on the basis of their size (although shape also plays a role). It can be used as either a preparative or an analytical technique.
SEC-MALS is a fast, powerful analytical technique for measuring the molecular weight of native proteins combining a size exclusion separation with a multiangle light scattering detector. If it is available to you, this method would be a very good way of studying the quaternary structure of your protein. It will tell you the mass of the complex. Combine this with SDS-PAGE, which will tell you about the number of different subunits and their masses. For relatively simple multimeric proteins, this may be enough information to infer the stoichiometry. For more complicated situations, you may need to quantify the amount of each subunit, which can be done by SDS-PAGE or size exclusion chromatography under strongly denaturing conditions.
Here is an article about FSEC and its uses.
http://www.chromatographyonline.com/fluorescence-detection-size-exclusion-chromatography-analytical-technique-multiple-applications-1
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