Some ligands/ small molecules solubility is pH- dependent. (E.g. biotin, EDTA)
Try adjusting the pH (it doesn't have to be accurate, just use a pH
paper if you have very small volumes). It might help you out to
improve ligand solubility (and binding with your protein).
Also, not to ruin the
I realised afterwards that this might be an experimental example of desperately
trying to turn a negative result into a positive one, which is something that
has been discussed here recently (sorry, Dale) ;-0 ;-0
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Original Message
On 24 Apr 2021,
If you want to try co-crystallisation again, if you dissolve your ligand in say
DMSO or anything that works, e.g. isopropanol, then add it to your protein up
to its maximum tolerable level (i.e. the level up to which the protein is not
denatured/inactivated by the solvent - you need to test
You may soak the crystals with ligands in maximum tolerable DMSO percentage
and harvest crystals at different time points. In my experience working
with some poor affinity, highly hydrophobic compounds, it may take up-to
several days for the ligand to bind with good occupancy (in one instance it
How long did you wait before freezing the crystal? Sometimes I have to wait
days before the ligand finds it way into the binding site.
Casper Wilkens
Asst. Prof.
Structural Enzymology & Biorefining
DTU Bioengineering
Technical University of Denmark
