AUTOMATED FERGUSON ANALYSIS OF GLYCOPROTEINS BY CAPILLARY ELECTROPHORESIS USING A REPLACEABLE SIEVING MATRIX

Obtaining accurate molecular weight estimates for glycoproteins by sod ium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been difficult due to the lack of SDS binding by the carbohydrate moie ties of the proteins. This leads to lower charge-to-mass ratios for SD S-glycoprotein complexes, resulting in over-estimation of molecular we ights by SDS-PAGE. In order to minimize these inaccuracies for protein s with abnormal charge-to-mass ratios, a Ferguson plot may be employed . This application requires the determination of relative mobilities f or standard proteins in addition to unknowns at several different gel concentrations. Historically, this technique has not been popular beca use it requires time-consuming preparation of gels with varying matrix concentrations, electrophoresis, and staining/destaining of gels. In this paper a procedure is demonstrated which automatically generates a ll of the data required for a Ferguson plot using a replaceable sievin g matrix (thereby eliminating gel polymerization) in a capillary forma t. In addition, this technique possesses the advantages inherent to ca pillary electrophoresis, namely, very fast separation times, and on-li ne monitoring which allows quantitation and precludes post-separation staining and destaining of gels.