Proteon xpr36 system

SPR was performed using a ProteOn™ XPR36 system (BioRad, Hemel Hempsted, UK). > 4000 RU of his-tagged PGK1 at concentrations of 80 μg/ml and 25 μg/ml was immobilized by amine coupling onto a GLH sensor chip. Subsequently, CRT0063465 was injected over the chip in a range of concentrations to determine K_d values. Purified recombinant PGK1 was obtained from Crelux (Martinsried, Germany).

The kinetics for chFVN145 binding with TBEV glycoprotein E was determined by a surface plasmon resonance (SPR) method using a ProteOn XPR36 System (Bio-Rad, USA). Recombinant protein rE [23 ] was immobilized onto vertical channel L1 of GLC sensor chip at a 70 response units (RU) level. Serial dilutions of chFVN145 were analyzed starting from the lowest concentration (1 nM, 3 nM, 9 nM, 27 nM, and 81 nM) at a flow rate of 25 μl/min. Vertical channel L2 was used as a reference channel. Binding experiments were performed in triplicate; chip surface was regenerated with 100 mM citric acid. Global analysis of experimental data based on a single-site or a heterogeneous analyte models was performed using the ProteOn Manager v. 3.1.0 software. The affinity constant was calculated as K_D = k_d/k_a.

To evaluate the target binding of the selected Z_MOMP affibodies to MOMP, surface plasmon resonance (SPR) was performed on a ProteOn XPR36 system (Bio-rad, CA, USA). The MOMP fusion protein (1 nM) served as the ligand was immobilized onto the surface of carboxylate glucans in HTG sensor chip (Bio-rad) and PBS was used as running buffer and for dilution of the analytes, as described previously (Zhu et al. 2020b (link); Xue et al.2016; Zhu et al. 2018 (link)). Subsequently, five or six concentrations of each affibody sample ranging from 125 to 4000 nM were prepared and injected over the chip surface to record sample binding to the surface. Z_WT affibody was set as a negative control. All experiments were carried out with a flow rate of 30 μL/min at 25 °C. The kinetic constants, including the association constant (ka), dissociation constant (kd), and affinity (KD, KD = kd/ka), were calculated by BIAcore T200 evaluation 3.0.2 software provided by the manufacturer, according to a 1:1 Langmuir binding model.

Surface plasmon resonance studies were performed on a ProteOn XPR36 system (Bio-Rad Laboratories). The running buffer was PBS-T-EDTA with 0.01% Tween 20. Human fragment-antigen binding binder (GE Healthcare, Chicago, IL, USA) was amine coupled to the surface of a GLM sensor chip (Bio-Rad Laboratories). BI-X (0.5 µg/mL) was captured on the surface. Twofold dilutions (starting at 10 nM) of Sema3A were injected over the surface. The resulting sensorgrams were fitted globally to 1:1 Langmuir binding (ProteOn Manager, version 3.1.0.6, Bio-Rad Laboratories) to provide values for on-rate (k_a), off-rate (k_d), and dissociation constant (K_D).

The binding affinity of EGCG for the two P. falciparum Hsp70s was determined using a Bio-Rad ProteOn XPR36 system (Hercules, CA, USA), as previously described [16 (link)]. Briefly, PfHsp70-1, PfHsp70-1_NBD, and PfHsp70-z (as ligands) were separately immobilized onto an HTE chip at concentrations of 0.5 μg/mL and 1 μg/mL, respectively, in order to validate the data. As analyte controls, ATP/ADP [16 (link)] were prepared at final concentrations of 0, 125, 250, 500, 1000, and 2000 nM, respectively, and were injected at 100 μL/min into each horizontal channel. In order to determine the association of EGCG with the respective ligand, the EGCG stock solution was prepared in deionised water. Aliquots of EGCG were prepared at final concentrations of 0, 125, 250, 500, 1000, and 2000 nM and injected at 100 μL/min into each horizontal channel. Association was allowed for 2 min and dissociation was monitored for 8 min. Data collected was double referenced using a buffer blank (buffer without EGCG) and a channel in which protein was excluded. Steady-state equilibrium constant data were processed and analysed using BioRad ProteOn Manager Version 3.1.0.6 (Hercules, CA, USA) and GE Healthcare Biosciences BIA evaluation Version 4.1.1 software (Uppsala, Sweden).

A Bio-Rad ProteOn XPR36 system was used to assess binding kinetics of the sdAb to target (L1 or SEB) immobilized on channels of a standard GLC sensor chip as detailed previously [18 (link), 19 (link)]. Briefly, the antigen was immobilized to the sensor chip surface on four rows at saturation concentrations of 10 µg/mL using EDC/NHS chemistry. The chip was turned 90 degrees, then dilutions of each sdAb (ranging from 100 to 0 nM) were flowed across the chip for 120 s at 100 µL/min, and the association was recorded. Next, dissociation was monitored as buffer was flowed over the chip for 600 s. The L1 surface was regenerated flowing through 50 mM glycine (pH 2.5) between individual samples. The one shot kinetics were determined from each of the antigen coated rows using five concentrations of single domain antibody, and kinetic parameters were calculated using the standard Langmuir binding model available on the ProteOn Manager RM 2.1 software (Bio-Rad). Typically the four values for K_D are within 20 %, and the range of values from the four measurements was always within a factor of 2.