Interaction of S100A6 with Target Proteins In Vitro and in Living Cells
S100A6 is a member of the EF-hand Ca2+-binding protein family, which plays important roles in a wide variety of Ca2+ signaling in the cells, as well as in pathophysiological conditions. Herein, we describe analytical protocols for evaluating the interaction of S100A6 with multiple target proteins in vitro, including biotinylated S100A6 overlay, glutathione-S-transferase (GST)-precipitation, surface plasmon resonance, and a GST-precipitation assay in living cells. These methods will elucidate the detailed molecular mechanisms of S100A6/target interactions and further improve our understanding of the physiological significance of S100A6-mediated Ca2+ signaling. Moreover, they may be used to evaluate other physical S100/target interactions.
Key wordsS100A6 Ca2+-binding EF-hand Protein–protein interaction FOR20 Protein phosphatase 5 GST-precipitation assay S100A6-overlay Transfection SPR analysis
This work was supported in part by a grant (to H.T.) from the Ryobi Teien Memory Foundation, Japan.