Hi everyone,
Still somewhat new to Stopped-Flow machines and fluorescence and wondering if there is an already-made solution to a potential problem.
I am working with a protein (HaloTag mutant D106A), which cannot form a covalent bond to its substrate (in these cases, a series of fluorescence dyes), but does have an initial association but then disassociates. Could I utilize fluorescence polarization on a Stopped-Flow machine to formulate what I suspect would be a curve (as the dye and protein reach an equilibrium of bond to unbound)? But from there, is there a way to calculate the disassociation rate (Kd, or 'off rate')? I have flipped through the manual (SX20-LED Stopped Flow Spectrometer from Applied Photophysics) but do not see anything answering this question.
If you are familiar with that software or the stopped-flow machine, am I missing that could help me calculate the Kd? Or could I do a protein titration (keeping dye constant) and just plot the plateau signal into something like Prism and do a non-linear curve to calculate the Kd? Any and all advice is appreciated!
Tom
The Kd is not the off-rate. It is the ratio of the off-rate constant k-1 to the on-rate constant k+1. Both of these constants can be measured by stopped flow. To measure k+1, mix the substrate and protein together rapidly and measure the rate of association as a function of the ligand or protein concentration. To measure k-1, premix the ligand and substrate, then rapidly dilute them and measure the rate of dissociation.
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