Purification and Characterization of iRFP713

iRFP713 and its variants in the holo- and apoforms were expressed and purified as described previously (Stepanenko et al., 2014 (link)) (Supplemental Methods 1). SDS/PAGE in a 12% polyacrylamide gels was used to confirm that the purity of the target proteins was at least 95% (Laemmli, 1970 (link)). The concentrated protein was stored in 20 mM Tris/HCl buffer, 150 mM NaCl, pH 8.0. The measurements were carried out at a low protein concentration (absorbance was kept less than 0.1, which corresponds to a protein concentration of 0.14 mg/ml) in 20 mM Tris/HCl buffer, pH 8.0, 1 mM tris(2-carboxyethyl)phosphine (TCEP).
GdnHCl, GTC, TCEP, and crowding agents PEG-8000, Dextran-40 and Dextran-70 were purchased from Sigma (St. Louis, MO, USA). The concentration of GdnHCl and GTC in stock solutions was calculated by the refraction coefficient measured by the Abbe refractometer (LOMO, St. Petersburg, Russia).