Measuring pH dependent kOFFs easily with the BLItz interferometer

Recently we published a new paper, describing the detailed effect of various histidines for the very important pH dependent release of HSP47 (a collagen chaperone) in the golgi (see here for more background).

The initial technical difficulty for this project was to determine reliably, small differences in kOFF values at different pH. Measuring kOFF values is trivial, using a BIACORE, however, as the kOFF goes into your resulting curve twice (once during association as kobs and once in the dissociation) you can’t directly compare the curves by eye and need to trust the curve fittings. Although this is exactly what we did in the final paper, earlier in this project we convinced ourselves by a different assay design. read more