Identification of proteins electroblotted to polyvinylidene difluoride membrane by combined amino acid analysis and...

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.