A procedure for purifying low-abundance protein components from the brain cytoskeleton-nuclear matrix fraction.
We describe a preparative procedure for low-abundance proteins of the cytoskeleton-nuclear matrix fraction from frozen bovine brain. Strigent centrifugation and washing conditions in the preparation of the cytoskeleton-nuclear matrix fraction are avoided to minimize loss of nuclear material. A recently described horizontal isoelectric focusing column, which tolerates appreciable precipitation, is used. In concert with selection of urea concentration and temperature, this isoelectric focusing apparatus provides a new approach to the fractionation of this complex, relatively insoluble mixture of proteins and other components. In addition, a heated, sodium dodecyl sulfate-sizing column has been utilized in order to eliminate interactions between the desired low abundance proteins and more abundant contaminating proteins. Together these procedures purify a specific low-abundance protein sufficiently to be detected by Coomassie blue staining in two-dimensional gels. The methods are robust and can be applied to multiple, relatively large brain samples (150 g of crude grey matter per batch); thus they should facilitate partial peptide sequencing for brain proteins of this operational class.
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0614.
K R
KR
Shelton
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E
Klann
G
G
Nixon
P M
PM
Egle
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002467:Cell Nucleus / Q000737:chemistry*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003599:Cytoskeleton / Q000737:chemistry*; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007531:Isoflurophate / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D012394:Rosaniline Dyes; D012711:Serum Albumin, Radio-Iodinated
D009419:Nerve Tissue Proteins; D012394:Rosaniline Dyes; D012711:Serum Albumin, Radio-Iodinated; D007531:Isoflurophate; C048139:Coomassie blue