Electrophoretic behavior of individual nuclear species as determined by capillary electrophoresis with laser-induced fluorescence detection.
We determined the feasibility of using capillary electrophoresis with postcolumn laser-induced fluorescence (CE-LIF) detection to characterize electrophoretic properties of isolated cell nuclei and impurities present in nuclear fractions. These fractions were isolated from NS-1 mouse hybridoma cells, stained with hexidium iodide, a DNA intercalating dye, and analyzed by CE-LIF detection. The corresponding electropherograms display two features: (i) broad peaks (6-90 s wide) caused by the cell culturing media and by free-DNA intercalated with hexidium iodide, and (ii) a large number of narrow peaks (180 ms wide), resulting from DNA associated with individual intact or disrupted nuclei. We confirmed that the narrow peaks were not caused by contaminating mitochondria. The overall electrophoretic mobility range of disrupted nuclei is 0 to -5 x 10(-4)cm(2)/Vs, while intact nuclei seem to have mobilities in the -1.5 to -3.5 x 10(-4)cm(2)/Vs range. Furthermore, the highly sensitive CE-LIF method reveals a high abundance of disrupted nuclei that cannot be directly observed by confocal microscopy.
Gunasekera
Nilhan
N
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Musier-Forsyth
Karin
K
Arriaga
Edgar
E
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000648:ultrastructure*; D019075:Electrophoresis, Capillary / Q000379:methods; D006825:Hybridomas / Q000166:cytology; D007834:Lasers; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods