A procedure for the extraction and high resolution two-dimensional gel electrophoresis of total nuclear phosphoproteins from isotonically purified nuclei.
Methods are described for the extraction and preparation of total nuclear proteins for high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The conditions for protein extraction and preparation limit both protease and phosphatase activity, allowing application of this technique to the reliable analysis of changes in nuclear protein composition and nuclear protein phosphorylation as well as other forms of post-translational modifications. Unlike other procedures for 2-D PAGE analysis of nuclear proteins the technique allows solubilization and extraction of all nuclear proteins along with removal of nucleic acids which interfere with isoelectric focusing and autoradiography of 32Pi-labeled proteins. It avoids lengthy dialysis in which precipitation of nuclear proteins often occurs and does not require precipitation and resolubilization of nuclear proteins to obtain sufficient protein concentrations for 2-D PAGE analysis; often impractical steps in which complete resolubilization of all proteins is not possible. It produces high resolution 2-D PAGE analysis in which identification of even low abundance proteins can be made, based on isoelectric point and molecular weight, allowing comparison with other studies.
Department of Biochemistry, West Virginia University Health Sciences Center North, Morgantown.
P C
PC
Baciu
J P
JP
Durham
D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002467:Cell Nucleus / Q000032:analysis*; D002999:Clone Cells; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007552:Isotonic Solutions; D009687:Nuclear Proteins / Q000302:isolation & purification*; D009696:Nucleic Acids / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification*; D012834:Silver; D013194:Staining and Labeling
D007552:Isotonic Solutions; D009687:Nuclear Proteins; D009696:Nucleic Acids; D010750:Phosphoproteins; D012834:Silver