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.
In the case of our protein interaction, the protein associates in the ER at around pH 7.5 and dissociates in the golgi at pH 6.0. So to simulate this behaviour we used a relative new device we had acquired: the BLItz.
As association is done in a 4µl drop holder and dissociation in a 500µl Eppendorf tube, it is very simple to change buffers in theses two phases. Additionally, buffer differences are not as problematic as in SPR, as the refractive index does not disturb the measurement.
So we observed association always at pH 7.5 and measured disscociation at different pHs (we used McIlvaine buffer as our buffer system). As you can see below these curves are strikingly different and you immediately see the difference between the wild-type and our found double mutant
Only these results encouraged to further investigate and make a “proper” association/dissociation analysis.