Improving SDS-PAGE method for monoclonal antibodies: The advantages of Tris-Acetate over Tris-Glycine SDS-PAGE system and comparison with CE-SDS method

Present study compares two different buffer systems for the electrophoretic separation of the IgG1 and IgG2 Monoclonal Antibodies using SDS-PAGE method. A modified Tris-acetate system was shown to be superior for separation of these proteins in a 6–20% gradient gel as compared with the traditionally used Tris-glycine method. This modified Tris-acetate buffer system showed sharper bands, more accurate determination of molecular weight, higher resolution, and better estimation of sub-fragments with closer results to those obtained by Capillary Gel Electrophoresis. Also in a parallel experiment, effect of IgG deglycosylation by PNGase-F enzyme was investigated and revealed no significant improvement on the SDS-PAGE results. •Tris-Glycine SDS-PAGE may not be suitable for large proteins like IgG.•Tris-Acetate SDS-PAGE with LDS sample buffer shows degraded forms similar to capillary gel electrophoresis results.•Tris-Glycine system does not show correct molecular weight of IgG and it is not related to protein glycosylation.•IgG2 samples appear as a one sharp band in Tris-acetate gels compared to smearing in Tris-glycine gels.•SDS-PAGE method is very simple and efficient method for primary evaluation of protein quality.