Notes: Nanoluciferase was used to characterize the conditions required to reliably perform single-cell BRET imaging of protein-protein interaction. HEK293T cells were cultured and transfected with vectors containing the Nluc gene sequence, such as is provided in the pNL1.1 Vector. Extracellular signal-related kinase (ERK) activity in neuronal dendritic spines, induced by the activation of endogenous synaptic NMDA receptors, was detected by an Nluc-optimized BRET-based sensor of ERK activity. The authors report that the use of Nanoluciferase improved the resolution in terms of both time and space, enhanced the duration of signal stability, expanded the dynamic range and increased the sensitivity of the BRET signals in single-cell imaging. (4691)

Notes: The pRL-CMV plasmid was used as an internal transfection efficiency control. The authors used a homebrew Renilla assay system. The buffer used was 25mM Tris (pH 7.5), 100mM NaCl, with 0.09µM coelenterazine (Biosynth) from an 0.09µM stock in 20mM HCl in methanol. (2170)