High performance density gradient electrophoresis of subcellular organelles, protein complexes and proteins

A density gradient electrophoresis (DGE) apparatus (2.2 Ø, 14 cm) was constructed for the rapid separation of milligram quantities of proteins. By using binary buffers according to Bier ( Electrophoresis 1993, 14 , 1011–1018) proteins were rate‐zonally separated in less than 60 min. Acidic proteins were separated in a pH 8.6, 56 μS/cm buffer, and basic proteins in a pH 5.4, 76 μS/cm buffer. Thus the A (p I 5.15) and B (p I 5.30) forms of β‐lactoglobulin as well as the sialylated glycoforms of apotransferrin were well separated at pH 8.6. The isoforms of myoglobin (p I 6.9 and 7.35, respectively), RNAse A (p I 9.45) and cytochrome c (p I 10.0) and lysozyme (p I 11) were separated at pH 5.4 within 80 min. On a 7 cm DGE column, subcellular organelles derived from HeLa cells were separated in standard electrophoresis buffer (655 μS/cm) for 90 min at 10 mA. Using a new low conductivity buffer (193 μS/cm) 20 min was sufficient to separate late endosomes, lysosomes, endoplasmic reticulum, early endosomes, plasma membrane, clathrin‐coated pits, proteasomes, and clathrincoated vesicles within a single run directly from a postnuclear supernatant.