| Assay Method Information | |
| | SPR Binding Assay |
| Description: | All binding assays were performed on a ProteOn XPR36 SPR Protein Interaction Array System (Bio-Rad Laboratories, Hercules, CA, USA). The instrument temperature was set at 25° C. for all kinetic analyses. ProteOn GLH sensor chips were preconditioned with two short pulses each (10 s) of 50 mM NaOH, 100 mM HCl, and 0.5% sodium dodecyl sulfide. Then the system was equilibrated with running buffer (1×PBS pH 7.4, 3% DMSO and 0.005% polysorbate 20). The surface of a GLH sensor chip was activated with a 1:100 dilution of a 1:1 mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.2 M) and sulfo-N-hydroxysuccinimide (0.05 M). Immediately after chip activation, the HIV-1 CA protein constructs were prepared at a concentration of 10 μg/mL in 10 mM sodium acetate, pH 5.5, and injected across ligand flow channels for 5 min at a flow rate of 30 μL/min. Then, after unreacted protein had been washed out, excess active ester groups on the sensor surface were capped by a 5 min injection of 1M ethanolamine HCl (pH 8.0) at a flow rate of 5 μL/min. A reference surface was similarly created by immobilizing a nonspecific protein (IgG b12 anti-HIV-1 gp120; was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Anti-HIV-1 gp120 Monoclonal (IgG1 b12) from Dr. Dennis Burton and Carlos Barbas) and was used as a background to correct nonspecific binding. Serial dilutions of hACSS-2 inhibitors were then prepared in the running buffer and injected at a flow rate of 100 μL/min, for a 50 s association phase, followed by up to a 5 min dissociation phase using the “one-shot kinetics” capability of the ProteOn instrument. Data were analyzed using the ProteOn Manager Software version 3.0 (Bio-Rad). The responses from the reference flow cell were subtracted to account for the nonspecific binding and injection artifacts. Experimental data were fitted to a simple 1:1 binding model. Experiments were performed in triplicate to detect kinetic and equilibrium dissociation constants (KD). |
| Affinity data for this assay | |
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