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A procedure for the extraction and high resolution two‐dimensional gel electrophoresis of total nuclear phosphoproteins from isotonically purified nuclei
Author(s) -
Baciu Peter C.,
Durham John P.
Publication year - 1990
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150110211
Subject(s) - isoelectric focusing , chemistry , isoelectric point , protein purification , chromatography , nucleic acid , polyacrylamide gel electrophoresis , nuclear protein , extraction (chemistry) , proteome , gel electrophoresis , biochemistry , enzyme , transcription factor , gene
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 solublization and extraction of all nuclear proteins along with removal of nucleic acids which interfere with isoelectric focusing and autoradiography of 32 P i ‐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.