Biacore t200 machine

The affinity of PBA toward α1-AT variants was measured by the fluorescence titration method where stock solutions of PBA were gradually titrated into α1-AT in phosphate-buffered saline with the fluorescence signal of the protein recorded at the 340-nm wavelength. The dissociation constant of the binding (K_d) was fitted with 1:1 binding mode by Origin software. The affinity of PBA toward α1-AT variants was also analyzed on a BIAcore T200 machine with CM7 chips (GE Healthcare). PBSP buffer (phosphate-buffered saline containing 0.005% P20) was used as the analysis buffer. α1-AT variants were immobilized on the chip through amine coupling chemistry, and PBA was diluted to concentrations ranging from 6.25 to 210 μm. PBA was flowed through the chip at a rate of 30 μl/min, and the response unit was measured. The sensor surface was regenerated with 10 mm glycine, pH 2.5, at the end of each cycle. Sensorgrams were fitted with BIAcore T200 analysis software using a 1:1 binding mode, and the K_d values were calculated with a steady-state affinity model due to the fast k_on and k_off (40 (link)).

Binding of BCR peptide to His-tagged RXRα-LBD was analysed at 25 °C by surface plasmon resonance (SPR) using a BIAcore T200 machine with CM5 chips (GE Healthcare). The purified His-tagged RXRα-LBD protein (20 μg ml⁻¹ in 10 mM sodium acetate, pH 5) in the presence of 9-cis-RA was immobilized on the CM5 chip via amine coupling of RXRα-LBD’s –NH₂ groups according to the manufacturer’s instructions. BCR peptide with different concentrations was injected into the chip. The sensor surface was regenerated with Glycine–HCl (10 mM, pH 2.5) when the data collection was finished in each cycle. Sensorgrams were fit globally with BIAcore T200 analysis using 1:1 Langmuir binding mode. The equilibrium dissociation constant (K_d) was determined using BIAcore’s evaluation software provided by the manufacturer.

The SPR experiments were performed using a Biacore T200 machine (GE Healthcare Life Sciences) at 20°C in 10 mM HEPES, pH 7.5, 150 mM NaCl, PtdIns(3,5)P₂ (C-35B6a), PtdIns(4,5)P₂ (C-45B6a), PtdIns(3,4,5)P₃ (C-39B6a) and Ins(1,3,4,5)P₄ (Q-1345) were purchased from Echelon Biosciences. To immobilize the biotinylated inositol phosphate onto the sensor chip, a BIAcore CM5 chip (GE Healthcare Life Sciences) was first derivatized with streptavidin following the manufacturer's instructions, and inositol phosphates were then injected on to channels 2 and 4 of the biosensor surface, leaving channels 1 and 3 as empty controls. The analyte with twofold serial dilutions was applied at a flow rate of 20 μl min⁻¹ for 180 s followed by 300 s of dissociation time. The biosensor chip was regenerated by 0.1% SDS after each running cycle. To perform the competition assay, the analyte (ASTN-2_701-1288) was incubated with a 10-fold molar concentration of Ins(1,3,4,5)P₄ and mannose-6-phosphate for 45 min, respectively, before being serially diluted in running buffer. The data were fit with the 1 : 1 Langmuir adsorption model (B = B_maxC/(K_d + C), where B is the response of bound analyte and C is the concentration of the analyte in the sample) to calculate the dissociation constant (K_d) using BIAcore BIAanalysis software.