Biacore t200 spr

The surface plasmon resonance (SPR) technology is a powerful tool for real-time monitoring of the interaction of binding kinetics and affinity. The SPR experiment was performed using the Biacore T200 SPR instrument (Biacore, GE Healthcare Life Sciences, Uppsala, Sweden) and sensor chip CM5 (carboxymethylated dextran surface, BR-1005-30, Cytiva). First, the protein Ywhaz was diluted to 30 μg/ml in immobilization buffer (10 mM NaAC pH = 4.0) and was immobilized via amine groups in the flow cell. The LWM was dissolved in DMSO (V900090, Sigma-Aldrich) and was diluted with 5% DMSO PBS-EP buffer solution successively to 500, 100, 50, 25, 12.5, 6.25, 3.125, and 50 μM. The sample was injected into the flow cell at a flow rate of 30 μl/min. The protein binding time was set to 120 s, and the dissociation time was 600 s. The chip was regenerated with glycine-HCl (pH 4.5, 10 mM). The data were retrieved and analyzed by Biacore T200 Evaluation Software, assuming the steady-state affinity analysis model.

The surface plasmon resonance (SPR) assay was conducted at 25°C using Biacore T200 SPR sensor (Biacore, GE Healthcare) with control software version 3.0. Briefly, a CM5 chip (carboxymethylated dextran surface) in Biacore PBS-EP running buffer was first activated following a standard EDC/NHS method. PTP1B protein was prepared in NaAc buffer (pH 4.5) to a final concentration of 20 μg/ml and was then injected into the channel of analysis instrument for 420 s followed by a 7 min injection of 1 M of ethanolamine buffer (pH 8.5) to block the residual active groups. For each sample analysis, another reference channel without the conjugated protein was also activated and blocked to eliminate non-specific binding to the surface of CM5 chip. Next, FA solution was diluted into different concentrations and injected into the analysis channel at a flow rate of 30 μL/min. The binding time of proteins and ligands was 420 s and the natural dissociation time was 420 s. All data were analyzed using Biacore T200 software.

The Omicron variant of SARS-CoV-2 SGP-RBD (B.1.1.529, Cat. # 40592-V08H121) was purchased from Sino Biological US, Inc (Wayne, PA). Based on the product information, Omicron variant of SARS-CoV-2 SGP-RBD protein construction: The DNA sequence encoding the SARS-CoV-2 Spike RBD (YP_009724390.1, with mutation G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H) (Arg319-Phe541) was expressed with a polyhistidine tag at the C-terminus. SPR measurements were performed on a Biacore T200 SPR or Biacore 3000 (Cytiva, Uppsala, Sweden). Streptavidin (SA) sensor chips and HBS-EP + buffer were purchased from Cytiva. Heparin was purchased from Celsus Laboratories (Cincinnati, OH). Heparin oligosaccharides, including hexasaccharide (degree of polymerization (dp)6), decasaccharide (dp10), tetradecasaccharide (dp14), and octadecasaccharide (dp18), and chemically modified heparins, including N-desulfated heparin, 2-O-desulfated IdoA heparin and 6-O-desulfated heparin, were purchased from Galen Laboratory Supplies (North Haven, CT).