Patent ID: 7795405

Claim:
A method of proteomic protein fractionation comprising: a) applying a protein mixture to an anion exchange column; b) collecting a first fraction comprising the nonbinding protein flowthrough that passes through the anion exchange column; c) eluting the anion exchange column with a high salt buffer to form an anion exchange eluate wherein the high salt buffer comprises 0.5 M salt or greater; d) applying the anion exchange eluate to a hydrophobic interaction chromatography column; e) collecting a second fraction comprising the nonbinding protein flowthrough that passes through the hydrophobic interaction chromatography column; f) eluting the hydrophobic interaction chromatography column with a low salt buffer, wherein the low salt buffer comprises 0.1 M salt or less, to form a low salt eluate; g) collecting a third fraction comprising the low salt eluate eluted from the hydrophobic interaction chromatography column; h) fractionating the first fraction by two-dimensional electrophoresis comprising isoelectric focusing and sodium dodecyl sulfate polyacrylamide electrophoresis on a first gel; i) fractionating the second fraction by two-dimensional electrophoresis comprising isoelectric focusing and sodium dodecyl sulfate polyacrylamide electrophoresis on a second gel; and j) fractionating the third fraction by two-dimensional electrophoresis comprising isoelectric focusing and sodium dodecyl sulfate polyacrylamide electrophoresis on a third gel, thereby generating more than 2 times the number of protein spots on the combination of the first gel, the second gel and the third gel, as compared to the number of spots on a single gel from a sample of the protein mixture that is not prefractionated by the anion exchange column and/or the hydrophobic interaction chromatography column, but is fractionated by two-dimensional electrophoresis comprising isoelectric focusing and polyacrylamide electrophoresis.