HIGH-RESOLUTION DENSITY GRADIENT ELECTROPHORESIS OF PROTEINS AND SUBCELLULAR ORGANELLES

Following a concept developed by Bier et al. (Electrophoresis 1993, 14 , 1011-1018), binary mixtures of amphoteric buffers with low conductiv ity and a good buffering capacity permit rapid rate zonal separation o f proteins on a density gradient electrophoresis apparatus (7 cm, O 2. 2 cm). At pH 8.66 and 250 V, beta-lactoglobulin (M-r 36 600) was separ ated into the A and B isoforms within 44 min; human transferrin (M-r 7 6 000-81 000) was separated into its sialylated glycoforms and carboni c anhydrase (M-r 30 000) separated into its isoenzymes. From these res ults we arrive at the term high-performance density gradient electroph oresis. Compartments belonging to the endosomal system were separated by density gradient electrophoresis. Early endosomes, recycling vesicl es, intermediate endosomes, late endosomes and lysomes became well-sep arated after 80 min at 10 mA using [I-125]transferrin and horseradish peroxidase as reporter molecules in pulse-cl ase regimes. Mixtures of Bier buffers and standard electrophoresis media permitted very short s eparation times (19 min at 10 mA) for the endosomal compartments. Conc ommittantly, endoplasmic reticulum and proteasomes were well resolved.