Imaging protein kinase C alpha activation in cells

Spatially resolved fluorescence resonance energy transfer (FRET) measured b y fluorescence lifetime imaging microscopy (FLIM), provides a method for tr acing the catalytic activity of fluorescently tagged proteins inside live c ell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase C alph a (PKC alpha) activation is identified and exploited. Activation of PKC alp ha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKC alpha by FLIM. This approach enabled the imaging of PKC alpha activation in live and fixed cultured cells and was also applied to pathological samples.