We describe a preparative procedure for low-abundance proteins of the cytoskeleton-nuclear matrix fraction from frozen bovine brain. Stringent 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.
- Frozen beef brain
- Heated sodium dodecyl sulfate column
- Low-abundance insoluble protein
- Preparative isoelectric focusing
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