Method and kit for identifying or characterizing polypeptides
AssigneeUniversity of Geneva
InventorsWilly Vincent Bienvenut, Denis Francois Hochstrasser, Jean-Charles Sanchez
Life of this patent
Estimating the term…
Abstract
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.
Claims21
A kit comprising:
- a) a first polypeptide-cleaving reagent suitable for incorporating in an electrophoretic gel on which at least one polypeptide has been isolated by electrophoresis, said first polypeptide-cleaving reagent being capable of cleaving said polypeptide to produce a partially cleaved polypeptide;
- b) at least one hydrophilic membrane capable of use in transblotting said partially cleaved polypeptide from said electrophoretic gel, the membrane having at least one second polypeptide-cleaving reagent immobilised thereon, said second cleaving reagent being capable of further cleavage of said partially cleaved polypeptide to produce polypeptide fragments;
- and
- c) a hydrophobic collection member suitable for receiving thereon said polypeptide fragments from said hydrophilic membrane when transferred thereto by transblotting.
A method of identifying or characterising polypeptides which have been isolated on a gel by electrophoresis, comprising the steps of:
- a) providing an electrophoretic gel on which at least one polypeptide has been isolated by electrophoresis;
- b) incorporating a first polypeptide-cleaving reagent in the gel, said cleaving reagent being capable of cleaving said isolated polypeptide contained by said gel to produce a partially cleaved polypeptide;
- c) providing adjacent to the gel at least one hydrophilic membrane on which is immobilised at least one second polypeptide-cleaving reagent capable of cleaving said partially cleaved polypeptide to produce polypeptide fragments;
- d) providing a hydrophobic collection member suitable for receiving thereon said fragments of polypeptide transferred thereto from said hydrophilic membrane by transblotting, said hydrophobic member being positioned beyond the hydrophilic membrane in a direction of movement of the fragments of polypeptide;
- e) partially cleaving the isolated polypeptide on the electrophoretic gel by the first polypeptide-cleaving reagent to produce a partially cleaved polypeptide, transblotting the partially cleaved polypeptide from the electrophoretic gel through the hydrophilic membrane under conditions effective to cause it to be further cleaved into polypeptide fragments by the second polypeptide-cleaving reagent, and transblotting the polypeptide fragments onto the hydrophobic collection member;
- and
- f) identifying or characterising the polypeptide fragments collected on the hydrophobic collection member.
Dependencies are inferred from the claim text.