Superose 12 pc 3.2 30 column

Gel filtration experiments on iRFP713 unfolded by GTC, GdnHCl, or urea were performed on a Superose 12 PC 3.2/30 column (GE Healthcare) using an AKTApurifier system (GE Healthcare). The samples of iRFP713 were prepared in buffer consisting of 50 mM NaH₂PO₄, 150 mM NaCl, pH 8.0 and containing the desired denaturant concentration. Protein samples were pre-incubated at 23 °C for 24 h. Ten microliters of the iRFP713 sample were then loaded on the column equilibrated with the same denaturant concentration. A set of proteins with known molecular mass (chromatography standards from GE Healthcare) was used for column calibration.

Absorption experiments were performed using a U-3900H spectrophotometer (Hitachi, Tokyo, Japan) with microcells 101.016-QS 5 mm × 5 mm (Hellma, Müllheim, Germany) at room temperature. The fluorescence spectra were recorded using a Cary Eclipse spectrofluorometer with 10 × 10 cells (Agilent Technologies, Mulgrave, Australia).
The extinction coefficient was determined by comparing the absorbance values at the far-red peak with the absorbance value at the Soret peak at about 390 nm. The extinction coefficient of tested proteins at the Soret band was supposed to be equal to that of free BV (39,900 M⁻¹·cm⁻¹). Nile blue dye was used as a standard for the evaluation of quantum yield. The chromophore binding with BphP1-FP and its variants was assayed by zinc-induced fluorescence and staining with Coomassie Blue of protein samples separated by SDS-PAGE [42 (link)].
Gel filtration experiments on BphP1-FP and its variants were performed on a Superose 12 PC 3.2/30 column (GE Healthcare, Chicago, IL, USA) using an AKTApurifier system (GE Healthcare). The samples of BphP1-FP and its variants were prepared in buffer consisting of 50 mM NaH₂PO₄, 150 mM NaCl, pH 8.0. Then 10 µL of protein sample was loaded on the column equilibrated with the same buffer. A set of molecular weight chromatography standards (GE Healthcare) were used for column calibration.

Gel filtration of sfGFP samples was performed on a Superose 12 PC 3.2/30 column (GE Healthcare) using an AKTApurifier system (GE Healthcare). Protein concentration was 0.4 mg/mL. The column was pre-equilibrated in native conditions (20 mM PBS, pH 7.4) or in denaturing conditions (6 M GdnHCl in 20 mM PBS, pH 7.4) before loading the appropriate sample. A set of proteins with known molecular mass (chromatography standards from GE Healthcare) was used for column calibration under native conditions.