Application of an improved density gradient electrophoresis apparatus to 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 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.
Tulp
A
A
The Netherlands Cancer Institute, Division of Cellular Biochemistry, Amsterdam.
Verwoerd
D
D
Pieters
J
J
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D004566:Electrodes; D004586:Electrophoresis / Q000379:methods*; D004912:Erythrocytes / Q000648:ultrastructure; D005362:Ficoll; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification; D008545:Melanoma / Q000648:ultrastructure; D008861:Microsomes / Q000648:ultrastructure; D015388:Organelles / Q000648:ultrastructure; D011506:Proteins / Q000302:isolation & purification*; D011817:Rabbits; D051381:Rats; D013347:Subcellular Fractions / Q000648:ultrastructure*; D014407:Tumor Cells, Cultured; D000515:alpha 1-Antitrypsin / Q000302:isolation & purification; D001619:beta-N-Acetylhexosaminidases / Q000302:isolation & purification
D007527:Isoenzymes; D011506:Proteins; D000515:alpha 1-Antitrypsin; D005362:Ficoll; D001619:beta-N-Acetylhexosaminidases