Instead of trying to find binding affinity yet, I just want to confirm whether a ligand binds my protein or not first.
I have a membrane protein to screen binding with a library of small organic molecules and I need to conserve protein stocks. I figured nano DSF is a good idea as a first pass until I decide which ligands are worth investigating in greater detail down the road.
What would be a good general protocol?
My thought was to go with something like I've shown in the picture and hope I see a thermal shift that indicates some kind of interaction.
Matthew van der Burg
Unlike traditional DSF methods, nanoDSF uses tryptophan or tyrosine fluorescence to track protein unfolding. Fluorescence intensity and fluorescence maximum are highly dependent on the chemical environment of tryptophan. So it all depends on the structure of your membrane protein. Check if there is tryptophan in the protein. I assume your method is suitable for the first try.
Yuri Mirgorod Hi Yuri, I have plenty of tryptophan residues.
Yeah, so my method is just a simple first-pass "bind or no-bind" at a small range of substrate concentrations. I wasn't entirely sure what concentrations to use but I figured 100uM, 5 mM, and 250 nM would give me a good binding range based on what I've seen in the literature and discussions on research gate. I don't know if I could just use a single substrate concentration instead of multiple because each substrate will have different affinities for the binding pocket.