Detection of tyrosine phosphorylated proteins by combination of immunoaffinity enrichment, two-dimensional difference gel electrophoresis and fluorescent Western blotting
2010 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 401, no 4, 581-585 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2010. Vol. 401, no 4, 581-585 p.
Fluorescence, Fluorescent Western blotting, Proteomics, Two-dimensional difference gel electrophoresis, Tyrosine phosphorylation
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:uu:diva-135372DOI: 10.1016/j.bbrc.2010.09.104ISI: 000283972400016OAI: oai:DiVA.org:uu-135372DiVA: diva2:374910