Apo–HasAp, GaSal–HasAp, and holo–HasAp were covalently bound to the surface of flow cells 2, 3, and 4 of a CM5 chip to a final level of 50 RU using the NHS-EDC kit (GE Life Sciences, Piscataway, New Jersey). Flow cell 1 was used as the blank. HasR-his (0–1000 nM) in 120 μL of HBS-EP buffer (GE Life Sciences) was injected into flow cells 1–4 at 25 °C until the signal reached saturation. The surface was then washed with the buffer for 3 min, and the dissociation of analyte–ligand complexes was followed over time. The flow cells were regenerated by injecting 15 μL aliquots of 10 mM glycine (pH 1.5) followed by 15 μL aliquots of 10 mM NaOH, and the process was repeated. Values from the reference flow cell were subtracted to obtain the values for specific binding. The maximum response units during the steady-state phase were plotted as a function of the HasR concentration, and data were fitted to a 1:1 binding model using BIAeval 4.1 software (Biacore).
Nhs edc kit
Manufactured by GE Healthcare
Sourced in United States
The NHS-EDC kit is a laboratory reagent used in the process of protein conjugation. It facilitates the covalent coupling of amine-containing molecules to carboxyl groups, enabling the creation of protein-ligand complexes. The kit provides the necessary chemicals and buffers required for this bioconjugation reaction.
Automatically generated - may contain errors
3 protocols using nhs edc kit
1
Kinetic Analysis of HasR Binding
2
Characterizing HasR-Holo-HasAp Binding Kinetics
3
Kinetic Analysis of SPINK1 Mutant Binding
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Kinetic analyses were performed using a Biacore T200 (GE Healthcare) at 25 °C. Amine coupling chemistry (NHS/EDC kit, GE Healthcare) was used to covalently immobilize SPINK1 and N34S mutant on a CM5 sensor chip surface (GE Healthcare). SPINK1 constructs were immobilized using a concentration of 10 μg/mL in 10 mM MES (2-(N-morpholino)ethanesulfonic acid) buffer, pH 5.5 to a density below 40 response units (RU). Ethanolamine-inactivated flow cells were used for referencing and data was collected at 10 Hz. Human cationic trypsin (MW: 26.5 kDa; GenWay Biotech, San Diego, US) was prepared as two-fold dilutions in running buffer (100 mM Tris, 150 mM NaCl, 1 mM CaCl2, 0.05% Tween20, pH 8.0 or 4.8, respectively). Trypsin was injected over the biosensor surface for 180 s followed by a dissociation time of 1000 s at a flow rate of 40 μL/min. The surfaces were regenerated after each cycle with regeneration solution (10 mM glycine/HCl, pH 2.0) for 100 s. Three independent experiments were carried out in each case. Data from six different concentrations between 1.56 and 50 nM were double referenced and fitted globally by a heterogeneous ligand model (BIAevaluation 3.1, GE Healthcare) to determine the binding parameters KD1,2, kon1,2 and koff1,2.
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