High-resolution density gradient electrophoresis of proteins and subcellular organelles.

Abstract

Following a concept developed by Bier et al. (Electrophoresis 1993, 14, 1011-1018), binary mixtures of amphoteric buffers with low conductivity and a good buffering capacity permit rapid rate zonal separation of proteins on a density gradient electrophoresis apparatus (7 cm, x 2.2 cm). At pH 8.66 and 250 V, beta-lactoglobulin (Mr 36600) was separated into the A and B isoforms within 44 min; human transferrin (Mr 76000-81000) was separated into its sialylated glycoforms and carbonic anhydrase (Mr 30000) separated into its isoenzymes. From these results we arrive at the term high-performance density gradient electrophoresis. Compartments belonging to the endosomal system were separated by density gradient electrophoresis. Early endosomes, recycling vesicles, intermediate endosomes, late endosomes and lysomes became well-separated after 80 min at 10 mA using [125I]transferrin and horseradish peroxidase as reporter molecules in pulse-chase regimes. Mixtures of Bier buffers and standard electrophoresis media permitted very short separation times (19 min at 10 mA) for the endosomal compartments. Concommittantly, endoplasmic reticulum and proteasomes were well resolved.