Grating Coupled Interferometry (GCI) - StructuralGenomicsConsortium/CNP25-CHIKV-nsP3-Macrodomain GitHub Wiki

C-terminally biotinylated and purified CHIKV Nsp3 Macrodomain (aa. 1334-1493) was immobilised onto a WAVE Creoptix PCH-STA Chip in Running Buffer: 10mM HEPES pH 7.2-7.5,150mM NaCl, 0.05% w/v Tween20, 0.5mM TCEP, 2.46% DMSO v/v [Nsp3] = 8.5µM f/c

The maximum increase in surface mass achievable was around 8000 pg/mm2 under the test conditions with gradual and continuous falling:

The rate of falling in surface mass after immobilisation will reduce overtime and eventually stabilise (fall rate however, will never reach 0)

Test Compounds (diluted in Running Buffer) were then injected in parallel into multiple flow channels over a surface that was immobilised with the protein (protein channel) and another surface that was not immobilised with protein (the reference channel). Change in surface density (refractive index) of both channel during the flow of compound were measured and subtracted. If increase in surface mass of the protein channel were significantly greater than that of the reference channel, it's treated as signal of binding. The gradual falling in surface mass of the protein-immobilised channel would lead to a falling baseline even after subtracting the reference channel signal (non-falling), this could be corrected by subtracting a blank (no compound, Running Buffer only) injection.