Premium
Microchip electrophoresis‐ SDS methods with high‐resolution and silver stain sensitivity for quality screening and quantitation of protein products
Author(s) -
Han Hongling,
Chen Xiaoyu
Publication year - 2012
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100511
Subject(s) - chromatography , chemistry , fluorophore , silver stain , resolution (logic) , fluorescence , reagent , sodium dodecyl sulfate , gel electrophoresis , electrophoresis , covalent bond , biochemistry , microbiology and biotechnology , physics , quantum mechanics , artificial intelligence , computer science , biology , organic chemistry
Two microchip electrophoresis ( ME )‐ SDS methods have been developed for high throughput quantitation and quality screening of protein products. Both methods utilize a commercial microchip instrument to separate dodecyl sulfate‐coated proteins within 1 min. In the high‐resolution ME‐SDS method, improved separation selectivity is achieved using a mixture of sieving polymers. Proteins of similar sizes, such as different fragment antigen‐binding (Fab) assemblies can be readily resolved and individually quantified. A high‐sensitivity ME ‐ SDS method was also developed with sensitivity comparable to that of SDS ‐ PAGE with silver staining. In this method, protein molecules are derivatized with a fluorescence reagent prior to analysis. LIF detection of the covalently attached fluorophore enables accurate quantitation of low‐expressing proteins and detection of minor species at 0.04% level (1 ng/mL loading concentration). Both the high‐resolution and the high‐sensitivity ME ‐ SDS methods can be applied to crude fermentation samples. The utilities of these methods in process development and formulation stability study are presented.