Application of an improved density gradient electrophoresis apparatus to the separation of proteins, cells and subcellular organelles

Abstract

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 constructed. Only 2.5 cm of the column were used for high resolution separations. A specially designed removable top cone permitted precise gradient introduction, thin sample layering (0.3-1 mm) and precise fractionation after electrophoresis. A bottom circular palladium anode (nongassing) was separated hydrodynamically but not electrically from the density gradient by a cation-permeable membrane. A top circular platinum cathode caused negatively charged particles to migrate upwards (levitation). Thin sample layering permitted short separation times (30-60 min) at only 3 V/cm (10 mA). As for proteins, glycoforms of a1-antitrypsin were separated as well as isoenzymes of beta-hexoseaminidase. Furthermore, separation of transferrin (Tf) from the putative Tf-receptor complex was effectuated. The device was equally suitable for the separation of Megadalton proteins (mucins). Artificial mixtures of intact erythrocytes (rat, rabbit, human) were separated with high resolution. About 10(7) cells (of 100 microns3 cell volume) could be loaded onto the device. Crude microsomes from the human melanoma cell line Mel JuSo were separated after brief trypsin treatment within 38 min at 10 mA. Ratios of the migration velocities of the constituent organelles were: late endosomes (LE):lysosomes (L):Golgi (G):early endosomes (EE) = 1:0.94:0.77:0.55 and under slightly different conditions LE:L:G:endoplasmatic reticulum (ER):plasma membrane (PM) = 1:0.87:0.64:0.58:0.49.

Survival of Pathogenic Mycobacteria in Macrophages Is Mediated through Autophosphorylation of Protein Kinase G

“…To independently confirm the results obtained by immunofluorescence microscopy, cell organelle electrophoresis was performed. During organelle electrophoresis, lysosomes become shifted in the electric gradient, whereas mycobacterial phagosomes remain in the unshifted position (7,12,29), allowing the physical separation of lysosomes and phagosomes. Cells infected with the mycobacterial strains indicated were homogenized, and total organelles were subjected to electrophoresis as described in Materials and Methods.…”

Inhibition of Phagosome Maturation by Mycobacteria Does Not Interfere with Presentation of Mycobacterial Antigens by MHC Molecules

“…To independently analyze the intracellular transport of wild-type M. bovis BCG or M. bovis BCG ⌬pkng in macrophages vs dendritic cells, subcellular fractionation by organelle electrophoresis was used (3,17,29). To that end, BM-macrophages and BMdendritic cells were infected with wild-type M. bovis BCG or M. bovis BCG ⌬pkng for 3 h, followed by homogenization and organelle separation by electrophoresis (Fig.…”

Generation of Specific Deoxynojirimycin-type Inhibitors of the Non-lysosomal Glucosylceramidase

“…Melanoma cells were cultured as described above, and a crude microsome fraction was prepared from a post-nuclear supernatant exactly as described earlier (13).…”

“…Glucosylceramidase-The easy access of hydrophobic AMP-DNM to the non-lysosomal glucosylceramidase in intact cells made us to look more closely into the localization of the enzyme using a recently developed subcellular fractionation technique that is a combination of density gradient centrifugation and free-flow electrophoresis (13,14). As shown in Fig.…”