Purification and Characterization of Fluorescent Proteins

All fluorescent protein coding sequences were inserted between BamHI and EcoRI sites in the constitutive expression vector pNCS which encodes an N-terminal 6×His tag and linker. Sequences for all primers used in this study are listed in Supplementary Table 4. Fluorescent proteins were expressed in E. coli strain NEBTurbo (New England Biolabs) or Mach1 (Invitrogen) by growing cultures in 2×YT medium supplemented with ampicillin overnight at 37 °C and shaking at 250 rpm. Fluorescent proteins were purified by Ni²⁺-affinity chromatography as previously described^{9 (link)}. Proteins were eluted in 50 mM Tris pH 7.5 or 50 mM sodium phosphate buffer pH 7.5 containing 250 mM imidazole. For all further characterization experiments, eluted fluorescent proteins were buffer-exchanged using Amicon Ultra0.5 10 kD MWCO ultrafiltration units (Millipore) into the same buffer without imidazole. Proteins were found to be stable when stored at 4°C indefinitely or when frozen at −20 °C or −80 °C.