ABSTRACT
This chapter provides a detailed protocol to assess the STIM/Orai interaction by fluorescence resonance energy transfer (FRET) and describes how to ensure the FRET assay is reliable and consistent and how to analyze and how to interpret the FRET data. It describes the new information on the molecular binding stoichiometry of the STIM1–Orai1 coupling interface. The physical interaction between STIM1 and Orai1 has been one of the most important parameters in order to understand the stoichiometry and gating mechanism of the STIM1/Orai1 complex. The molecular nature of the coupling interaction between the activated STIM1 protein and the Orai1 channel remains elusive. The STIM and Orai coupling interaction are particularly suited to quantitative analysis by FRET. Before any FRET imaging measurements are taken, the instrument must be calibrated to determine the following parameters: ratio of fluorescence intensity for equimolar donor and acceptor used, and bleed-through coefficients for donor and acceptor.
