title: Identification of proteins electroblotted to polyvinylidene difluoride membrane by combined amino acid analysis and bioinformatics: An ABRF multicenter study.
abstract: The ABRF amino acid analysis study evaluated the general utility of amino acid analysis (AAA) for identification of proteins after denaturing gel electrophoresis and electroblotting to polyvinylidene difluoride (PVDF) membrane.Thirty-eight participating laboratories analyzed a known control (ovalbumin, 5 microg applied to the gel) and either lysozyme or bovine serum albumin as unknown samples (1-, 5-, and 10-microg amounts applied to the gel). Analyses of the unknowns yielded average compositional errors of approximately 30%, 19%, and 18%, respectively, from the low, intermediate, and higher sample amounts; the ovalbumin control exhibited an approximately 17% average error. Compositional data were submitted to the ExPASy and PROPSEARCH Internet sites for protein identification.Without search parameter adjustments or restrictions, both computer programs provided identification of about 20%, 66%, and 74% of the data from the 1-, 5-, and 10-microg gel samples, respectively. Deleting problematic data (Gly, Met, and Pro) did not always facilitate protein identification. Incorporating control results into the ExPASy search increased identifications 2% to 10%, and restricting search parameters by species, isoelectric pH, and molecular weight increased identifications by more than 80%. Average amounts analyzed for correct identifications were approximately 0.4 microg, 1.8 microg, and 2.9 microg for the 1-, 5-, and 10-microg gel samples, respectively.The results support the efficacy of AAA in the low microgram and nanogram range for the identification of PVDF-immobilized proteins from two-dimensional gels.
authors.affiliation: Biochemisches Institut, University of Zurich, Zurich, Switzerland. Peter.Hunziker@access.unizh.ch
authors.forename: P
authors.initials: P
authors.lastname: Hunziker
authors.forename: T T
authors.initials: TT
authors.lastname: Andersen
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authors.lastname: Bao
authors.forename: S A
authors.initials: SA
authors.lastname: Cohen
authors.forename: N D
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authors.lastname: Denslow
authors.forename: J D
authors.initials: JD
authors.lastname: Hulmes
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authors.initials: AM
authors.lastname: Mahrenholz
authors.forename: K
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authors.lastname: Mann
authors.forename: K M
authors.initials: KM
authors.lastname: Schegg
authors.forename: K A
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authors.lastname: West
authors.forename: J W
authors.initials: JW
authors.lastname: Crabb