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.

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.

Abstract: 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 “sandwich”. 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.