Rapid isolation of rat brain nuclei on percoll gradients.
A rapid isotonic method for fractionation of nuclei from rat brain is described. This procedure is based on the use of discontinuous colloidal silica gel (Percoll) gradients. We start from a 63,000-g purified nuclear pellet (fraction P3) isolated from gray matter and white matter separately. This is followed by fractionation of fraction P3 in an initial differential centrifugation step on five-step Percoll gradients producing six nuclear fractions designated 1, 2, 3 (gray matter) and 4, 5, 6 (white matter). Fractions 2, 4, and 5 obtained from this centrifugation are heterogeneous. These fractions are subfractionated further under isopycnic conditions using five-step Percoll gradients to yield subfractions 2b, 4b, and 5c. Three methods were used to characterize the nuclear types. First, light and electron microscopic examination was used to identify the nuclei in each preparation and to assess the purity of each preparation. Second, the activities of RNA polymerase I and II were monitored. Third, the protein/DNA ratios of the nuclear fractions were determined. Fraction 1 was enriched in neuronal nuclei; fractions 2b and 4b in astrocytic nuclei; and fractions 3, 5c, and 6 in nuclei of oligodendrocytes. RNA polymerase I and II activity was highest in fraction 1, which also displayed the highest protein/DNA ratio. Electron microscopy showed that the various classes of nuclei are congruent to 90% pure. Therefore, the procedure described here is suitable for obtaining highly purified neuronal and three types of glial nuclei from rat brain.
Vakakis
N
N
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Hearn
M T
MT
Veitch
B
B
Austin
L
L
D000818:Animals; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure*; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000032:analysis / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011205:Povidone; D051381:Rats; D011919:Rats, Inbred Strains; D012822:Silicon Dioxide
D003902:Detergents; D009419:Nerve Tissue Proteins; C016039:Percoll; D012822:Silicon Dioxide; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D011205:Povidone