Glycine hcl

All the reagents used in SPR experiments were purchased from GE Healthcare, including HBS‐P+ (Buffer 10×; BR‐1006‐71), amine coupling kit (BR100050), EDC (0.4 M), NHS (0.1 M) and ethanolamine hydrochloride (1 M; pH 8.5). For regeneration, the following regeneration buffers were tested (GE Healthcare): NaOH (50 mM; BR‐1003‐58) and glycine/HCl (10 mM; pH 2.5; BR‐1003‐56); acetate 5.0 (BR‐1003‐51) was used for coupling. In order to regenerate the SPR chip following binding with lectins, the following elution solutions were used (Vector Laboratories): that is, solutions for eluting mannose/glucose‐binding lectins (ES‐1100‐100); galactose/GalNAc‐binding lectins (ES‐2100‐100); fucose/arabinose‐binding lectins (ES‐3100‐100); GlcNAc/chitin‐binding lectins (ES‐5100‐100) and sialic acid‐binding lectins (ES‐7100‐100).
SPR assays were run on Biacore X100 (GE Healthcare) using a sensor chip CM5 (29‐1496‐04) under a constant flow rate of 30 μl/min at 25°C. Original SW Biacore X100 Control Software was used to operate the instrument.

Surface plasmon resonance (SPR) assays were performed on a Biacore 3000 instrument (GE Healthcare, Uppsala, Sweden). We used a heparin-coated SPR sensor chip (Heparin Approx. 50 nm hydrogel chip, XanTec bioanalytics GmbH, Dusseldorf, Germany) for assaying the binding characteristics between different AT mutants and heparin. Wild-type and mutant AT were diluted in running buffer (HEPES 10 mM, NaCl 150 mM, EDTA 3 mM, surfactant 0.005% (v/v), pH 8.4) and injected over the heparin-coated sensor chip surface at 6 different concentrations (40, 80, 120, 160, 200 and 240 nM) at a flow rate of 10 μL/min for 7 min. Between two measurements, sensor chip surfaces were regenerated with 30 μL regeneration buffer (10 mM glycine-HCl, pH 2.5, GE Healthcare, Uppsala, Sweden). The optimal pH of the interaction was previously determined; thus, the analysis proceeded at pH 8.4 [43 (link)]. A Langmuir 1:1 binding model was used for curve fitting. The rate constants for association and dissociation (ka and kd) as well as the equilibrium constants for association and dissociation (KA and KD) were calculated from the sensorgrams. BIAevaluation software version 3.2 (GE Healthcare, Uppsala, Sweden) was used to evaluate the interaction analysis.

Binding competition between mAbs 7B3, 1C11, and 6A6 and five other EDIII-targeted antibodies was determined using a real-time, label-free, bio-layer interferometry assay on an Octet RED96 biosensor (ForteBio, Fremont, CA) as previously described [24 (link)]. The experiment was performed at 30°C in PBS buffer with shaking at 1,000 rpm. Ni-NTA biosensors (ForteBio) were first loaded with 4 μg/mL His-tagged-E protein for 300 s and then associated with the first mAb (7B3, 7F4, 8D10, 1C11, 6A6, 6B6, 6D6, or 6F1) for 900 s. An irrelevant mAb, 2D1, was used as negative control and PBS was used as blank solution. The biosensors were then dipped into the second mAb and incubated in the presence of the first mAb. The capacity of additional binding was monitored by measuring further shifts for 300 s. All mAbs were evaluated at concentration of 150 nM, except for 6B6 (700 nM), 6D6 (900 nM), and 6F1 (900 nM), for saturation measurement. The Ni-NTA biosensors were regenerated with 10 mM glycine-HCl (pH 1.7; GE Healthcare) and re-charged with 10 mM NiCl₂. The response of mAb binding to the E protein was compared and the data were processed using BIAevaluation software (Biacore, Uppsala, Sweden).

Tropoelastin (purified from chicken aorta) was purchased from Elastin Product Co. (Owensville, MO). Sensor chip CM5, sodium acetate buffer, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS), ethanolamine and glycine-HCl were purchased from GE Healthcare (Marlborough, MA). Rabbit anti-GST IgG antibodies conjugated with HRP was purchased from GenScript (Piscataway, NJ). HRP-conjugated goat anti-hamster IgG antibody and 3,3′,5,5′-tetramethylbenzidine (TMB) peroxidase substrate were purchased from Kirkegaard and Perry Laboratories (Gaithersburg, MD). Polyclonal antibodies specifically against L. biflexa serovar Patoc ligA were generated by immunizing hamsters with the same spirochetes twice, and the anti-sera were collected from hamsters 1 week after second immunization. Hamsters were used under conformity of animal protocols, which were approved by Cornell University Institutional Animal Care and Use Committee (IACUC, Protocol number: 2015-0133). Animals were cared for in adherence to the policies of the NIH Office of Laboratory Animal Welfare (OLAW), the standards of the Animal Welfare Act, the Public Health Service Policy, and the Guide for the Care and Use of Laboratory Animals.

Kinetic binding parameters for the interaction of human ACVR2B.Fc fusion protein with recombinant Activin A and Activin A muteins were determined under neutral pH on Biacore 3000 using dextran-coated (CM5) chips at 25°C. The running buffer was prepared using filtered HBS-EP (10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, 0.05% polysorbate 20, pH 7.4). The capture sensor surface was prepared by covalently immobilizing Protein A (Sigma-Aldrich, St. Louis, MO) to the chip surface using (1¬Ethyl-3-[3-dimethylaminopropyl]carbodiimide-hydrochloride)/N-hydroxysuccinimide (EDC/NHS) coupling chemistry. Binding kinetics of Activin A and Activin A muteins were measured by flowing 100nM-0.14 nM of ligand, serially diluted three-fold, at 100 uL/minute for two minutes and monitored for dissociation for 15 min. All capture surfaces were regenerated with one 30 s pulse of 10 mM glycine–HCl (pH 1.5, (GE Healthcare, Marlborough, MA). Kinetic parameters were obtained by globally fitting the data to a 1:1 binding model with mass transport limitation using Scrubber 2.0 c Evaluation Software. The equilibrium dissociation constant (KD) was calculated by dividing the dissociation rate constant (kd) by the association rate constant (ka).

Fishing experiments were performed on a BIACORE T200 instrument at 25°C. FGFR1 protein was immobilized on all four channels of a CM5 chip following a standard EDC/NHS protocol. JQ-R tablets (JQ-R is a mixture of refined extracts from Coptis chinensis, Astragalus membranaceus, and Lonicera japonica, the three main constituents of JQJTT) were fully dissolved in water, and the insoluble residue was pelleted by centrifugation and discarded. Then, the supernatant was then injected at a flow rate of 10 μl/min. All four flow cells were used for analyte capture and recovery. The bound material was eluted with 0.5% trifluoroacetic acid.
For the kinetics experiment, six or seven analytes were serially diluted twofold, prepared in the running buffer, and injected at a flow rate of 30 μl/min onto the CM5 chips, followed by 1 min of dissociation data acquisition. To obtain more extensive off-rate decay data, an additional injection of the analyte with the third-highest concentration was included in each experiment. All covalent surfaces were regenerated with 10 mM glycine–HCl (pH 2.5, GE HealthcaMolecular Dockingre). All experiments were conducted at 25°C.