University of Geneva

Willy Vincent Bienvenut

Denis Francois Hochstrasser

Jean-Charles Sanchez

Polypeptides which have been separated by gel electrophoresis can be identified or characterized by a procedure which has two main stages. In the first stage the gel is digested with a polypeptide-cleaving agent such as an enzyme. This produces mainly large fragments which, in the second stage are electroblotted through a hydrophilic membrane on which is immobilized another polypeptide-cleaving reagent such as an enzyme onto a hydrophobic member, typically a membrane, e.g. of PVDF. The resulting fragments, usually peptides, are identified, preferably by MALDI-TOF MS, or a property may be determined, e.g. by interaction with an antibody.

Method and kit for identifying or characterizing polypeptides

Method of identifying polypeptides

Polypeptides separated on a gel are identified by cleaving the separated polypeptides with an immobilized cleaving reagent such as an enzyme, and transferring the fragments to a hydrophobic collection layer where they are analyzed. A hydrophilic membrane containing an immobilized protease such as trypsin is provided between an electrophoresis gel and a hydrophobic membrane to form an electroblotting &ldquo;sandwich&rdquo;. Polypeptides separated on the gel by electrophoresis are electroblotted from the gel through the hydrophilic membrane where they are cleaved by the protease into fragments, and the fragments are collected on the hydrophobic membrane where they are identified such as by MALDI-TOF MS analysis. From identification of the fragments, the polypeptide from which they came is identified. The hydrophilic membrane may be provided with functional groups to which the protease is immobilized by covalent bonding. Free residual functional groups not bound to the protease are capped to prevent their reaction with the polypeptide. The functional groups may be activated carbonyl groups, carboxylic acid groups or carboxylic acid derivative groups capable of reacting with an amino group of an enzyme. A kit for use in carrying out the electroblotting is formed containing the cleaving reagent immobilized on the hydrophilic membrane, and the hydrophobic collection layer. The immobilized cleaving reagent and hydrophobic layer may be in separate containers or in the same container.

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Thenac, France

Willy Vincent Bienvenut