title: Detection of tyrosine phosphorylated proteins by combination of immunoaffinity enrichment, two-dimensional difference gel electrophoresis and fluorescent Western blotting.
abstract: We describe fluorescence-based 2-D gel electrophoresis methods for visualization of low abundant, cancer relevant tyrosine phosphorylated (pTyr) proteins. The methods investigated were fluorescent Western blotting and two-dimensional difference gel electrophoresis (2-D DIGE) for detection of non-enriched and immunoaffinity enriched pTyr protein patterns. The same anti-phosphotyrosine specific antibody, 4G10, was used for both approaches. The results from fluorescent Western blotting of total proteins and from enriched CyDye DIGE pre-labeled pTyr proteins showed similar down regulation of phosphorylation upon treating of cells from a cancer model system (K562 chronic myeloid leukemia cells) with imatinib. This treatment introduced a known perturbation of phosphorylation that enabled testing of these new approaches to analyze variations in tyrosine phosphorylation levels. Enrichment of pTyr proteins was found highly advantageous for the outcome. Out of a simplified 2-D DIGE experiment of immunoaffinity enriched control and treated pTyr proteins, differential analysis as well as protein identification by mass spectrometry (MS) was possible.
authors.affiliation: Uppsala University, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-75185 Uppsala, Sweden. Sara.Lind@genpat.uu.se
authors.forename: Sara
authors.initials: S
authors.lastname: Bergström Lind
authors.forename: Sa
authors.initials: S
authors.lastname: Hagner-McWhirter
authors.forename: Lioudmila
authors.initials: L
authors.lastname: Elfineh
authors.forename: Magnus
authors.initials: M
authors.lastname: Molin
authors.forename: Anneli
authors.initials: A
authors.lastname: Jorsback
authors.forename: Johan
authors.initials: J
authors.lastname: Ohman
authors.forename: Ulf
authors.initials: U
authors.lastname: Pettersson
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005453:Fluorescence; D006801:Humans; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis*; D014443:Tyrosine / Q000032:analysis*
chemical_list: D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine