Density gradient electrophoresis of cells in a reversible gel.
Density gradient electrophoresis permits the separation of cell types according to surface charge density with high resolution. Any source of flow compromises the resolving power of density gradient electrophoresis. Although procedures have been devised to successfully counteract electroosmotic and convective flows, the final collection of separands requires that they be pumped out of the electrophoresis column. Experiments were therefore designed to test the hypothesis that this flow could also be eliminated by trapping the separated bands in a gel, from which they could be collected by slicing the gel cylinder. Glutaraldehyde-fixed rat and rabbit erythrocytes were used as test particles in a phosphate-buffered isotonic Ficoll-sucrose density gradient in a 2.2 cm diameter, thermostated vertical glass column that could be opened at both ends. Two types of agarose were used as gel polymers: Electrophoresis grade agarose (J.T. Baker Chemical Co.) at final concentrations of 0.1 to 0.25% and SeaPrep ultralow gelling agarose (Marine Colloids Div., FMC Corp.) at a final concentration of 1.0%. Electrophoretic separability of the test particles and fluid stability were tested independently at 55 degrees C and 32 degrees C at which the two agaroses were, respectively, liquid. The experiments demonstrated that the higher temperatures required and the presence of agarose compromised neither the stability of the density gradient nor the migration properties of the cells, and cells can be separated in a sol at a temperature that is compatible with cell viability.
Plank
L D
LD
Althouse Laboratory, Pennsylvania State University, University Park.
Kunze
M E
ME
Gaines
R A
RA
Todd
P
P
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D003911:Dextrans; D004586:Electrophoresis / Q000379:methods*; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation / Q000379:methods*; D004912:Erythrocytes; D011817:Rabbits; D051381:Rats
D003911:Dextrans; C025614:sephadex