TomkUCL
commented
7 months ago Attempt 1: 13th March 2024
OPERATORS: @jelenaUCL @TomkUCL
PROTEIN PRODUCTION: @jelenaUCL Pegha Ghiabi at SGCToronto.
RESULTS: 20240314_GCI_nsp13_test.pptx
EXPERIMENT DESIGN:
For reference on use of this assay, see https://doi.org/10.1038/s41598-020-79226-w .
The binding kinetics of Nsp13 were measured by the label-free, high-sensitivity GCI WAVE instrument (Creoptix AG, Wädenswil, Switzerland). The experiments were conducted employing specific optical sensor chips, the so-called WAVEchips (Creoptix AG) (see Fig. 1). To analyse the amount of immobilised Nsp13 protein, a WAVEchip with a functional PCH-SA coating was used. This type of sensor chip can be used for capturing ligands through specific and directional biotin-streptavidin (SA) interactions. The PCH chip carries a thick polycarboxylate-based hydrogel coating with free carboxylic functions to which proteins can be covalently coupled through EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysuccinimide) linking chemistry.
Owing to their multiple-channel design, WAVEchips allow for referencing the signal recorded on the target channel with a signal measured on a selected reference channel. In our experimental design, we immobilized Nsp13 to the target channel (Ch3), whilst no protein was captured on the reference channel (Ch1). The handling of all the different solutions including sample pickup, delivery and injection onto the channels was performed fully automatically by a robotic autosampler (WAVEsampler). Throughout the entire experimental period HBS-EP 10 mM HEPES, 150 mM NaCl, pH 7.4 was used as running buffer (RB).
The basic element of a GCI experiment is the cycle, which consists of a baseline (running buffer injection), a sample (e.g., protein solution) injection as well as a washing section (running buffer injection). A typical GCI experiment can be separated into a sequence of series, where a series is usually composed of more cycles.
As a first experimental step, the polycarboxylate layer of the 4PCH-STA WAVEchip was conditioned using a solution of 0.1 M sodium tetraborate decahydrate (borate), 1 M NaCl, pH 9.0, which was followed by sequential running buffer injections (so-called start-up cycles). In the immobilisation step, the avi-tagged Nsp13 protein (12.7 mg/mL Nsp13 solution dissolved in the running buffer) was immobilised to the chip surface on Ch3 by physisorption of biotin molecule on the streptavidin anchoring groups were injected into Ch3. The control of the device as well as data adjustment and kinetic analysis were all performed using the WAVE-control software (Creoptix AG). The raw kinetic data were adjusted using the processed data and were fitted using the “heterogeneous ligand” kinetic model of the WAVEcontrol software (for the model equations, see ...
Compounds were screened in HBS-EP buffer with 2% DMSO at 8 concentrations following a 3-fold dilution series.
Please refer to issue #25 for previous discussions using surface-plasmon resonance. This issue describes updated efforts at establishing a robust biophysical screen to confirm binders to the SARS-CoV-2 helicase, Nsp13, including a viable positive control compound.