High-resolution two-dimensional electrophoresis of nuclear proteins: a comparison of HeLa nuclei prepared by three different methods.
A comparative analysis of HeLa cell nuclear proteins is presented using Iso-Dalt methods of protein resolution in two dimensions. The nuclear proteins were prepared by (1) spin through glycerol cushion, (2) spin through sucrose cushion, or (3) Triton wash. Improved resolution of total nuclear proteins in the range of pH 4.5-6.0 was achieved by substituting longer isotubes in combination with broad-range ampholines during the isoelectric focusing step. An attempt to indicate silver stainable protein spots common to total cellular extracts and nuclear preparations has been made. Also, proteins that appear to be well represented in all three nuclear preparations and remain undetectable in the total cellular protein pattern have been marked as probably being enriched nuclear proteins. Such a comparative analysis of whole nuclear protein preparations made it possible to document that the different preparations preserved the same set of proteins. The Triton-wash method of obtaining nuclei was identified as the preferred choice. Coomassie-stained gels and blots of these nuclear proteins could serve as a guide for accessing relevant protein spots for further biochemical analysis such as immunoblotting.
Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007.
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Prasad
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Dritschilo
D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000302:isolation & purification; D002467:Cell Nucleus / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D007526:Isoelectric Point; D009363:Neoplasm Proteins / Q000302:isolation & purification; D009687:Nuclear Proteins / Q000302:isolation & purification*; D018809:Proliferating Cell Nuclear Antigen; D016760:Proto-Oncogene Proteins c-fos / Q000302:isolation & purification; D016755:Proto-Oncogene Proteins c-jun / Q000302:isolation & purification
D000951:Antigens, Neoplasm; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun