Akta fplc upc 900 system

Analysis was performed using the AKTA UPC-900 FPLC system (GE Healthcare) with a Superdex 200 10/200GL column, with the following parameters: sample volume 500 µl, eluent buffer 20 mM Tris-HCl, 100 mM NaCl, pH 7.2, flow rate 0.5 ml/min.

All ¹H-NMR spectra are reported in parts per million (ppm) and were measured relative to the signals of DMSO (2.5 ppm). ¹³C-NMR spectra are reported in ppm relative to those of residual DMSO (40 ppm). The mass spectra of the chemicals were obtained using a Waters LC–MS instrument equipped with a single-quadrupole mass detector and an electrospray ionization source (Waters ACQUITY QDa), while those of the proteins were obtained using Agilent 6100 equipment with a series of triple-quadrupole mass spectrometers (Agilent Technologies, CA, USA). Protein purification was performed using AKTA UPC 900 FPLC system (GE Healthcare), and absorption spectra were obtained at room temperature using a UV–visible spectrometer (Agilent 8453, Agilent Technologies). Fluorescence spectra were obtained using a microplate reader equipped with SkanIt 2.4.3 RE software for Varioskan Flash (Varioskan Flash, Thermo Fisher Scientific Inc.). Cell fluorescent images were recorded at room temperature by confocal and fluorescence microscopy (FV1000, OLYMPUS).

Synthetic genes, with optimized E. coli codons, of IgFLNa21 domain (Gly²²³⁶-Ser²³²⁹) and hybrid IgFLNa21 domain (P⁷⁵²LFKSATTTVMN⁷⁶³-GASGSGASGS-G²²³⁶-S²³²⁹ were sub cloned into pET24a and pET14b vectors (Novagene), respectively. Transformed E. coli rosetta cells (Merck) were grown at 37 °C in rich media (LB) or supplemented M9 minimal media containing either ¹⁵N ammonium chloride and/or ¹³C glucose (Cambridge Isotope Laboratories). Protein production was induced by addition of 0.5 mM IPTG at OD₆₀₀ ~ 0.6–0.7 and cells were further grown at 20 °C for 10–12 hours. Cells were harvested through centrifugation and pellets were resuspended in buffer A (50 mM sodium phosphate buffer and 300 mM NaCl, pH 8.0) followed by cell lysis using sonication. Lysates were loaded onto Ni-NTA column (GE) for His-tag based affinity purification using AKTA FPLC UPC-900 system (GE Healthcare UK Ltd., England). His-tagged proteins were first eluted using buffer B (50 mM sodium phosphate buffer, 500 mM imidazole, and 300 mM NaCl, pH 8.0), then exchanged into buffer C (20 mM sodium phosphate buffer and 50 mM NaCl, pH 6.0). Protein samples were further purified by size-exclusion chromatography in buffer C using a Superdex 200 10/300 column with a flow rate of 0.3 ml/min. NMR samples were prepared, through buffer exchange, in buffer D (25 mM sodium phosphate buffer, 5 mM NaCl, 2 mM DTT and 10% D₂O, pH 6.5).