i like this post (click again to cancel)
i dont like this post (click again to cancel) remove favorite mark from this question (click again to restore mark)

I am trying to setup an experiment using HSQC with titration of a compound to determine the Kd of the protein-ligand interaction. My difficulties have been in the sample preparation/formulation, as these novel compounds are very weakly soluble in aqueous buffers. I must be able to quantify the soluble concentration of compound over ~6-8 points to generate the binding curve (signal intensity/chemical shift vs. [L]).

Previous observation of this protein by HSQC and other NMR techniques have used a buffer containing 5 mM HEPES, 1 mM DTT, 5 mM EDTA in 90% H2O/10% D2O, pH 7.5. Compounds have not been soluble in this buffer, and only slightly more so in this buffer + 5% DMSO.

In each of the following methods of solubilization, compounds were successfully dissolved in an organic solvent, but delivery into an aqueous buffer suitable for the protein have yielded opaque solutions with precipitate material.

(a) Dissolve dry compound in DMSO, then add small volume of compound stock to NMR buffer.

(b) Dissolve dry compound in CHCl3, deposit on glass vial surface, then add buffer and sonicate and/or place in ultrasonifer water bath.

(c) Dissolve dry compound in concentrated HCl, then add buffer drop-wise.

I would appreciate any answer to the question of how to prepare a sample using weakly soluble compounds such that I can calculate the Kd of the protein-ligand binding.

asked Jul 31 '12 at 07:37

Patrick%20Bartlow's gravatar image

Patrick Bartlow

One Answer:
i like this answer (click again to cancel)
i dont like this answer (click again to cancel)

I know a commercial platform that provides custom protein services, such as crystallization, structure determination and function analysis by X-ray, EM, NMR, etc. They provide MagHelix™ NMR platform based on spectrometers ranging from 300 MHz to 800 MHz and Saturation Transfer Differences (STD) NMR service. NMR (STD-NMR) has been widely used to characterize ligand-receptor complexes for many years and STD NMR has been used to monitor ligand binding to proteins.


answered Jun 29 at 19:39

Justin%20Frank's gravatar image

Justin Frank

Your answer
Please start posting your answer anonymously - your answer will be saved within the current session and published after you log in or create a new account. Please try to give a good answer, for discussions, please use comments and please do remember to vote (login to vote)
toggle preview

powered by CNPROG