Purification and Characterization of GCaMP Proteins

GCaMPs in pRSETa were transformed into chemically competent BL21(DE3)-pLysS, and purified via the N-terminal His tag. Protein concentration was determined by intrinsic tryptophan fluorescence. Calcium clamping was performed at pH 7.2 with 10 mM blends of K₂H₂EGTA and Ca₂EGTA from the Calcium Calibration Kit #1 (Invitrogen). Free [Ca²⁺] levels were calculated using MAXCHELATOR (maxchelator.stanford.edu). Fluorescence spectra were recorded on a Safire2 (link) fluorescence plate reader (Tecan). The dynamic range here is calculated as F_max/F_min. F_max is the fluorescence intensity at saturating [Ca ²⁺] and F_min is the fluorescence intensity at zero [Ca ²⁺].