Affinity Purification of GO-Matryoshka Biosensors from for Quantitative Ratiometric Fluorescence Analyses.

Genetically encoded biosensors are powerful tools for quantitative visualization of ions and metabolites . Design and optimization of such biosensors typically require analyses of large numbers of variants. Sensor properties determined such as substrate specificity, affinity, response range, dynamic range, and signal-to-noise ratio are important for evaluating data. This protocol provides a robust methodology for binding assays of newly designed sensors. Here we present a detailed protocol for purification and characterization of genetically encoded sensors, exemplified for the His affinity-tagged GO-(Green-Orange) MatryoshCaMP6s calcium sensor. GO-Matryoshka sensors are based on single-step insertion of a cassette containing two nested fluorescent proteins, circularly permutated fluorescent green FP (cpGFP) and Large Stoke Shift LSSmOrange, within the binding protein of interest, producing ratiometric sensors that exploit the analyte-triggered change in fluorescence of a cpGFP.