Procedure For The Fractionation Of Proteins By Using Sequential Ion Exchange And Hydrophobic Interaction Chromatography As Prefractionation Steps Before Analysis By Two Dimensional Electrophoresis
AssigneeGuild Associates, Inc.
InventorAugustine DiNovo
Life of this patent
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Abstract
After the sequencing of the human genome, great interest has developed in trying to discern the complementary proteome of humans and other species. The present disclosure provides devices, systems, and methods for proteomic fractionation that may increase the number of protein spots visualized by 2DE analysis, and may allow enrichment of proteins normally not detectable by standard 2DE analysis. According to some embodiments of the disclosure, devices, systems, and methods of the disclosure relate to fractionating a proteome on the basis of surface charge, hydrophobicity, isoelectric point and/or size.
Claims23
A method of proteomic protein fractionation comprising:
- applying a protein mixture to an anion exchange column,
- eluting the anion exchange column with a high salt buffer to form an anion exchange eluate,
- applying the anion exchange eluate to a hydrophobic interaction chromatography column,
- eluting the hydrophobic interaction chromatography column with a low salt buffer to form a low salt eluate, and
- fractionating the low salt eluate by isoelectric focusing and polyacrylamide electrophoresis.
A method of proteomic protein fractionation comprising:
- prefractionating a protein mixture using a first binary chromatographic mode to form a no-bind mode 1 fraction and a bind mode 1 fraction;
- prefractionating a bind mode 1 fraction using a second binary chromatographic mode to form a bind mode 1, no-bind mode 2 fraction and a bind mode 1, bind mode 2 fraction;
- and
- fractionating a bind mode 1, bind mode 2 fraction by isoelectric focusing and polyacrylamide electrophoresis to form at least two gel fractions.
A method of proteomic protein fractionation comprising of:
- applying a protein mixture comprising up to every protein in an organism's proteome to an anion exchange column,
- eluting the anion exchange column with a high salt buffer without a gradient to form an anion exchange eluate,
- applying the anion exchange eluate to a hydrophobic interaction chromatography column,
- eluting the hydrophobic interaction chromatography column with a low salt buffer without a gradient to form a low salt eluate,
- eluting the hydrophobic interaction chromatography column with a low salt buffer comprising an organic solvent to form a low salt plus solvent eluate,
- fractionating the low salt eluate by isoelectric focusing and polyacrylamide electrophoresis, and
- fractionating the low salt plus solvent eluate by isoelectric focusing and polyacrylamide electrophoresis.
A method of proteomic protein fractionation comprising:
- applying a protein mixture to an anion exchange column under conditions that permit formation of a high pI fraction,
- eluting the anion exchange column with a high salt buffer to form an anion exchange eluate,
- applying the anion exchange eluate to a hydrophobic interaction chromatography column under conditions wherein a hydrophilic fraction is formed,
- eluting the hydrophobic interaction chromatography column with a low salt buffer to form a low salt eluate,
- fractionating the high pI fraction by isoelectric focusing and polyacrylamide electrophoresis,
- fractionating the hydrophilic fraction by isoelectric focusing and polyacrylamide electrophoresis, and
- fractionating the low salt eluate by isoelectric focusing and polyacrylamide electrophoresis.
Dependencies are inferred from the claim text.