Molecular weight standards for calibration of gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Ferritin and apoferritin

Abstract

Ferritin and apoferritin are widely used for the calibration of gel filtration columns and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and are commercially offered for these purposes as part of molecular weight calibration kits. Many of the reported applications are severely in error as presented in leading references and application manuals. The manufacturers have based their recommendations on incorrect physicochemical parameters in the literature and incorrect or inadmissible assumptions about the protein subunit composition and architecture and have not taken into account the unusual resistance of these proteins to denaturation in SDS. Here the relevant physicochemical parameters of horse spleen apoferritin as reported in the literature are critically reevaluated and the best current estimates are identified fied as the following: weight average molecular weight of apoferritin. M w = 481.200 : molecular weight of subunits, major subunit, M L = 19,889: minor subunit, M H = 22,200: apparent specific volumes in 0.02 m acetate buffer, pH 5.5, and 0.1 m NaCl, φ ∗ = 0.721 mlg 1 and φ′ = 0.743 mlg −1; partial specific volume at 20°C, v ̄ = 0.738 mlg −1 ; viscosimetric molar volume, M[ η] = 1.78 × 10 6ml mol −1; Stokes radius, R St = 67.1 A ̊ ; viscosimetric radius, R vis = 65.6 A ̊ ; sedimentation coefficient s 20,w o = 16.6 S; translational diffusion coefficient, D 20,w = 3.24 × 10 7cm 2 s 1. Recommendations are provided for proper application of ferritin and apoferritin for calibration purposes in gel filtration and SDS-polyacrylamide gel electrophoresis.