APPLICATION OF AN IMPROVED DENSITY GRADIENT ELECTROPHORESIS APPARATUSTO THE SEPARATION OF PROTEINS, CELLS AND SUBCELLULAR ORGANELLES

A DGE apparatus, made of Perspex, consisting of a separation column (5 X 2.2 cm) and containing a 0-4% linear Ficoll density gradient, was c onstructed. Only 2.5 cm of the column were used for high resolution se parations. A specially designed removable top cone permitted precise g radient introduction, thin sample layering (0.3-1 mm) and precise frac tionation after electrophoresis. A bottom circular palladium anode (no ngassing) was separated hydrodynamically but not electrically from the density gradient by a cation-permeable membrane. A top circular plati num cathode caused negatively charged particles to migrate upwards (le vitation). Thin sample layering permitted short separation times (30-6 0 min) at only 3 V/cm (10 mA). As for proteins, glycoforms of alpha 1- antitrypsin were separated as well as isoenzymes of beta-hexoseaminida se. Furthermore, separation of transferrin (Tf) from the putative Tf-r eceptor complex was effectuated. The device was equally suitable for t he separation of Megadalton proteins (mucins). Artificial mixtures of intact erythrocytes (rat, rabbit, human) were separated with high reso lution. About 10(7) cells (of 100 mu m(3) cell volume) could be loaded onto the device. Crude microsomes from the human melanoma cell line M el JuSo were separated after brief trypsin treatment within 38 min at 10 mA. Ratios of the migration velocities of the constituent organelle s were: late endosomes (LE) : lysesomes (L) : Golgi (G) : early endoso mes (EE) = 1 : 0.94 : 0.77 : 0.55 and under slightly different conditi ons LE : L : G : endoplasmatic reticulum (ER) : plasma membrane (PM) = 1 : 0.87 : 0.64 : 0.58 : 0.49