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Identification of enzymes and activity from two-dimensional gel electrophoresis.
Nat Protoc
January 2008
Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18, 1090 Vienna, Austria.
Identification of proteins with enzymatic activity by mass spectrometry (MS) and concomitant determination of function by screening enzyme activity from two-dimensional gel electrophoresis (2DE) is one of the challenges of gel-based proteomics. In this protocol, proteins are extracted from spinal cord tissue followed by 2DE with in-gel digestion and identification by matrix-assisted laser desorption/ionization. Protein spots identified as possible enzyme of interest are punched, eluted by SDS-containing Tris buffer and renatured by buffers under reductive conditions.
View Article and Find Full Text PDFAn immunochemical micromethod was designed to estimate total IgG and IgG sub-classes of anti-insulin antibodies in immunized diabetic patients. Insulin, immobilized on a solid phase, was allowed to react with serum samples containing anti-insulin antibodies. Bound anti-insulin IgG interacted with mouse monoclonal antibodies specific for total IgG or for each IgG isotype.
View Article and Find Full Text PDFThe immunosorbent micromethod with the use of nitrocellulose paper was employed for the first time to demonstrate subviral antigens from animal origin, such as the M-protein of the Newcastle Disease virus. The method proved to be highly effective. It was useful in discovering amounts as high as 10-20 ng of the antigen, while with the use of the enzyme-linked immunosorbent assay (ELISA) amounts of about 200 ng could be evaluated.
View Article and Find Full Text PDFImmunochemical methods are based on the antigen-antibody reaction. Two groups of techniques can be made: group I in which the visualization of the antigen-antibody reaction is direct (radial immunodiffusion, immunonephelometry, immunoturbidimetry..
View Article and Find Full Text PDFAntisera have been prepared against purified proteoglycan monomer and link protein from bovine cartilages. The immunological properties of these species have been studied by immunodiffusion, Laurell rocket immunoelectrophoresis, crossed immunoelectrophoresis, hemagglutination, and immunofluorescence. Antisera raised against either adult bovine articular or foetal bovine epiphyseal proteoglycan monomer were monospecific.
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