An improved method to obtain a soluble nuclear fraction from embryonic brain tissue.
This paper describes modifications of the standard methods for obtaining a soluble nuclear fraction from embryonic brain tissue. The main improvements are: (1) the inclusion of a low speed centrifugation step to prevent the appearance of high density contaminants, (2) a sucrose density gradient to remove perinuclear mitochondria and ER membranes and (3) a protein extraction approach which significantly enhances protein yield. To demonstrate the effectiveness of the method, pellets were analyzed by light and electron microscopy and purity of the soluble extracts was immunologically tested. Finally, to illustrate the applicability of this approach, the induction of the transcription factor HIF-1 (hypoxia-inducible factor-1) was assessed by Western blot using soluble nuclear fractions and by immuno-electron microscopy using purified nuclear fractions, both obtained from the optic lobes of chick embryos. In conclusion, the procedure presently described appears to be reliable and convenient for obtaining a pure soluble nuclear fraction from a discrete amount of embryonic brain tissue.
Giusti
Sebastián
S
Institute of Cell Biology and Neuroscience "Prof. E De Robertis", University of Buenos Aires, Paraguay, Argentina.
Bogetti
María Eugenia
ME
Bonafina
Antonela
A
Fiszer de Plazas
Sara
S
D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000737:chemistry / Q000648:ultrastructure; D002498:Centrifugation; D002642:Chick Embryo; D051793:Hypoxia-Inducible Factor 1 / Q000096:biosynthesis; D016253:Microscopy, Immunoelectron; D009687:Nuclear Proteins / Q000302:isolation & purification; D017830:Octoxynol; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D013501:Surface-Active Agents
D051793:Hypoxia-Inducible Factor 1; D009687:Nuclear Proteins; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D017830:Octoxynol