Protein kinase fractionation and enrichment by multicolumn affinity chromatography of lysate from A549 cells. Protein kinase fractionation and enrichment.

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Protein kinase fractionation and enrichment by multicolumn affinity chromatography of lysate from A549 cells. Protein kinase fractionation and enrichment by multicolumn affinity chromatography of lysate from A549 cells. To visualize the proteins specifically enriched by the different inhibitor affinity, 2% of the elution fractions from the bisX, AX14596, PP58, and purB columns were compared with 0.0004% aliquots of the total lysate and flow-through by SDS gel electrophoresis and silver staining (upper panels). In parallel, 0.3% of the elution fractions from each specific column elution were separated together with 0.02% aliquots of the lysate and flow-through. After transfer to nitrocellulose membrane, immunoblotting was performed with specific antibodies recognizing GSK3, RIPK2, p38, and PAK4 as indicated. CSFR, colony-stimulating factor receptor; EGFR, epidermal growth factor receptor; Eph, ephrin receptor; FAK, focal adhesion kinase; FGFR, fibroblast growth factor receptor; FRK, Fos-regulatory kinase; InsR, insulin receptor; IRR, insulin receptor-related; JAK, janus kinase; PDGFR, platelet-derivid growth factor receptor; ROR, regeneron orphan receptor; SYK, spleen tyrosine kinase; TIE, tyrosine kinase with immunoglobin and EGF, repeats. Josef Wissing et al. Mol Cell Proteomics 2007;6:537-547 © 2007 The American Society for Biochemistry and Molecular Biology