HIGH-PERFORMANCE DENSITY GRADIENT ELECTROPHORESIS OF SUBCELLULAR ORGANELLES, PROTEIN COMPLEXES AND PROTEINS

A density gradient electrophoresis (DGE) apparatus (2.2 empty set, 14 cm) was constructed for the rapid separation of milligram quantities o f proteins. By using binary buffers according to Bier (Electrophoresis 1993, 14, 1011-1018) proteins were rate-zonally separated in less tha n 60 min. Acidic proteins were separated in a pH 8.6, 56 mu S/cm buffe r, and basic proteins in a pH 5.4. 76 mu S/cm buffer. Thus the A (pI 5 .15) and B (pI 5.30) forms of beta-lactoglobulin as well as the sialyl ated glycoforms of apotransferrin were well separated at pH 8.6. The i soforms of myoglobin (pl 6.9 and 7.35, respectively), RNAse A (pl 9.35 ) and cytochrome c (pI 10.0) and lysozyme (pI 11) were separated at pH 5.4 within 80 min. On a 7 cm DGE column, subcellular organelles deriv ed from HeLa cells were separated in standard electrophoresis buffer ( 655 mu S/cm) for 90 min at 10 mA. Using a new low conductivity buffer (193 mu S/cm) 20 min was sufficient to separate late endosomes, lysoso mes, endoplasmic reticulum, early endosomes, plasma membrane, clathrin -coated pits, proteasomes, and clathrin-coated vesicles within a singl e run directly from a postnuclear supernatant.