Method of identifying polypeptides
AssigneeUniversity of Geneva
InventorsWilly Vincent Bienvenut, Denis Francois Hochstrasser
Life of this patent
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Abstract
Polypeptides which have been separated by gel electrophoresis can be identified by electroblotting them through a “sandwich” comprising in order: a) the separation gel; b) at least one hydrophilic membrane, e.g. of carboxyl-modified PVDF, on which is immobilized at least one reagent capable of cleaving a polypeptide, e.g. trypsin; c) a hydrophobic layer, typically a membrane, e.g. of PVDF. Preferably a biased alternating current or discontinuous direct current is used for electroblotting. The resulting fragments, usually peptides, are identified, preferably by MALDI-TOF MS.
Claims9
A method of identifying polypeptides which have been separated by gel electrophoresis on the same gel, comprising:
- a. providing adjacent to the gel a hydrophilic membrane on which is immobilized a cleaving reagent capable of cleaving a polypeptide, wherein the reagent generates fragments of the separated polypeptides;
- b. providing a hydrophobic collection member suitable for receiving thereon the fragments of polypeptide transferred thereto by electroblotting, wherein said member is a film, membrane or support having a hydrophobic layer thereon, said hydrophobic member being positioned beyond the hydrophilic membrane in a direction of movement of the fragments of polypeptide;
- c. electroblotting the polypeptides through the hydrophilic membrane, under conditions wherein the cleaving reagent cleaves the polypeptides into fragments which are collected on the hydrophobic member;
- and
- d. identifying the fragments collected on the hydrophobic member, wherein identification of the fragments, identifies the polypeptide from which they came.
A method of identifying polypeptides which have been separated by gel electrophoresis on the same gel, comprising:
- a. providing adjacent to the gel a hydrophilic membrane on which is immobilized an enzyme capable of cleaving a polypeptide, wherein the enzyme generates fragments of the separated polypeptides;
- b. providing a hydrophobic collection member suitable for receiving thereon the fragments of polypeptide transferred thereto by electroblotting, wherein said member is a film, membrane or support having a hydrophobic layer thereon, said hydrophobic member being positioned beyond the hydrophilic membrane in a direction of movement of the polypeptide fragments;
- c. electroblotting the polypeptides from the separation gel through the hydrophilic membrane, under conditions wherein the enzyme cleaves the polypeptides into fragments which are collected on the hydrophobic member;
- and
- d. identifying the fragments collected on the hydrophobic membrane wherein identification of the fragments identifies the polypeptides from which they came.
Dependencies are inferred from the claim text.