Biacore cm5 chip

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.

SPR experiments were performed using a Biacore T200 machine (GE Healthcare Life Sciences) at 20°C in 10 mM HEPES (pH 7.5) and 150 mM NaCl. PtdIns(4,5)P₂ (C-45B6a) and PtdIns(3,4,5)P₃ (C-39B6a) were purchased from Echelon Biosciences. The BIAcore CM5 chip (GE Healthcare Life Sciences) was firstly covered with streptavidin following the manufacturer's instructions before the biotinylated PIPs or biotinylated proteins were immobilised. The analyte with twofold serial dilutions was applied at a flow rate of 20 µl/min for 180 s followed by 600 s of dissociation time. The biosensor chip was regenerated after each sample injection cycle with a different buffer in the two experimental set-ups: in the case of immobilised PIPs on the sensor chip, 0.1% SDS was used; when the PH domain was immobilised, then the surface was regenerated by 2 M MgCl₂. The data were fit with a 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 the BIAanalysis software. The molecular mass of the nanodiscs for SPR analysis was estimated to be 120 kDa.

For sdAb binding and affinity ranking against different recombinant HA a BIAcore T100 machine (GE Healthcare) was used in combination with a single cycle kinetics procedure [55 (link)]. In brief, purified recombinant HA from H1N1 strains between 1918 and 2010 (A/South Carolina/1/1918, A/New Caledonia/20/1999, A/Solomon Islands/3/2006, A/Brisbane/59/2007, A/California/07/2009, A/Christchurch/16/2010) and H5N1 subtype A/Vietnam/1194/2004 (Protein Sciences) were immobilised onto a BIAcore CM5 chip in 10mM sodium acetate pH 5.5 using an amine coupling kit (GE Healthcare). A concentration series of purified sdAbs was sequentially run over the different antigen surfaces ranging from 1nM to 10nM. A reference surface was subtracted prior to evaluation of the sensograms using the single cycle kinetics procedure of the BIAevaluation software (GE Healthcare) in combination with a 1:1 fitting model.