Hispur ni nta spin plates

BL21-Gold (DE3) cells transformed with plasmids containing computationally-designed or evolved RFP variants were used to inoculate deep 24-well plates (Whatman) containing 5 mL of Overnight Express Instant TB medium (Novagen) supplemented with 100 μg mL⁻¹ ampicillin (pET11-a) or kanamycin (pET29b(+)). These plates were sealed with sterile and pierced silicone sealing mats (Axygen) and incubated overnight at 37 °C with shaking. Cells were then harvested by centrifugation and washed twice with phosphate buffer pH 7.4. Pellets were stored at 4 °C to allow for chromophore maturation. After two days, cell pellets were resuspended in 400 μL of lysis buffer (50 mM sodium phosphate buffer, pH 8.0, 300 mM sodium chloride, 2.5 mM imidazole, 1× Bugbuster protein extraction reagent [Novagen], 1 mg mL⁻¹ hen egg white lysozyme [Sigma], 10 U Benzonase nuclease [Novagen]) and plates were incubated at 25 °C with gentle shaking for 20 minutes. After incubation, clarified lysates were collected by centrifugation and proteins were purified using HisPur Ni-NTA spin plates (Thermo Scientific) according to the manufacturer's protocol. Protein purity was verified by SDS-PAGE. These proteins were used for initial spectroscopic characterization (see below).