Abstract
Polyacrylamide gel electrophoresis (PAGE) is routinely used for electrophoretic characterization of biomolecules. In this study we demonstrate that this technique is quite useful for the characterization of small molecules, however, it becomes inadequate for the resolution of highβmolecularβweight macromolecules like MUC2 mucins. We show that agarose gel electrophoresis (AGE) constitutes a better medium through which MUC2 macromolecules can penetrate and hence be resolved. MUC2 molecules obtained from PC/AA (a benign colonic adenoma cell line) were extracted, and their molecular weights (varying between 15β32βΓβ10^6^ Da) determined using Laser Light Scattering (LLS). These mucin molecules were reduced until 5β6βΓβ10^6^ subunits were obtained. The reduced and unreduced mucin molecules were run on an AGE and Western blotting and visualized, but were too large for PAGE. For this reason, the mucin subunits were treated with trypsin to obtain 500β600 kDa fragments. These tryptic fragments were then run on PAGE and AGE, and their resolutions compared. We conclude that AGE is a more useful electrophoretic method for characterization of macromolecules like mucin glycoproteins than PAGE.
Agarose gel electrophoresis of PC/AA cell layer extract (U: unreduced, intact form, R: reduced form).
magnified imageAgarose gel electrophoresis of PC/AA cell layer extract (U: unreduced, intact form, R: reduced form).