The EYFP mutants were generated using IVA-cloning PCR technique [26] (link) with the following oligonucleotide set containing the appropriate substitutions (primer sequences are shown in Suppl. methods ). pQE30 plasmid vector backbone (Qiagen) and E. coli XL1 Blue strain (Evrogen) were used for the DNA constructs assembly.
FP variants, cloned into the pQE30 vector (Qiagen) with a 6His tag at the N-terminus, were expressed in E. coli XL1 Blue strain (Evrogen). The proteins were isolated by ultrasonic cell lysis, purified using TALON metal-affinity resin (Clontech), eluted from the adsorbing material by treatment with 100 mM imidazole (pH 8) and then desalted by ultrafiltration with Amicon® Ultra-0.5 10 K (Merck) filters. All isolation and chromatography procedures were performed in PBS buffer pH 7.4 (Gibco). The same solution was further used for the specimen storage and spectroscopy measurements.
FP variants, cloned into the pQE30 vector (Qiagen) with a 6His tag at the N-terminus, were expressed in E. coli XL1 Blue strain (Evrogen). The proteins were isolated by ultrasonic cell lysis, purified using TALON metal-affinity resin (Clontech), eluted from the adsorbing material by treatment with 100 mM imidazole (pH 8) and then desalted by ultrafiltration with Amicon® Ultra-0.5 10 K (Merck) filters. All isolation and chromatography procedures were performed in PBS buffer pH 7.4 (Gibco). The same solution was further used for the specimen storage and spectroscopy measurements.