I am not aware of any consensus regarding ligand dilution in your choice of buffer ( in your case, buffer B). However, from my own experience, compounds dissolved in dmso, diluted about 1:50-1:100 times in B gives consistent and reproducible results. For example, if your total reaction volume is 200ul then I would be reluctant in adding anything more than 4 ul of your compound stock

Sometimes though, you may not be able to make compound parent stocks at high enough concentrations either due to high cost of the item limiting the amount you purchase or because you cannot dissolve the required amount in your solvent of choice as the compound precipitates, signifying you have overshot the saturating concentration.

In these cases, you can add up to 10 ul of the compound. Be cautious of your experimental design and include rigorous controls. I would not go above that as it could affect protein structure , cause an immiscible bilayer ( drastically interferes with solution homogeneity) and reduce compound interaction with your protein of interest.

All the best for your experiments.

Hi!

What about the final pH of the solution. Is there any study that generalises the maximum percentage of ligand that can be employed without causing any change in the pH of the final solution?

No general percentage: you just have to make sure that the vehicle (anything in the ligand solution except the ligand itself) does not interfere with the assay.

For pH, that depends on the buffer capacity of your two solutions, for organic solvents, it depends on the sensitivity of your protein to organic solvents. For example, if your protein and your ligand are dissolved in the same vehicle, you can combine them at any ratio, but if some component in your ligand solution can interfere with your assay, the concentration of that component in the reaction mix has to be kept at a level that does not interfere.

Dear Devanshu Mehta,

building on what Annemarie Honegger just said, there are several buffer systems that allow you to buffer your solutions in a wide range of pH, not restricting yourself to the "narrow" window of pKa.

When we need to do SPR or ITC at two very different pH's, we quite often use these new buffer systems that allow us to have one solution (just an example), at pH 4.3 and the other at pH 8.9 IN THE SAME BUFFER.

You can find some information here: https://www.moleculardimensions.com/products/c323-Special-Buffers

Best,

Jose

@ Devanshu : no , it depends on what solution you employ to dissolve the ligand. As a general rule, adding at the dilution I mentioned above seems to work , at least for me , and the articles I have read that study protein ligand behaviour. There will always be a minor change in the pH of your protein buffer solution but that is usually negligible and doesn't cause changes to your assay.

Thank you everyone for the help.

There are several different problems here:

Thank you Engelbert Buxbaum

Dear Devanshu,

you may want to take a look at Figure 2 of this reference in DDT journal

https://reader.elsevier.com/reader/sd/pii/S1359644601017809?token=9222390EA70B5E5B311A3EF2F57658A778E4B2E667E8AB094E795E4130AFB3F17BB6086F3549E0F3437CC3C509F145AA

I do not know whether your question relates to NMR based work. In NMR you will ALWAYS have a influence of pH or organic solvent on position of NMR signals. I recommend to acquire data of the target protein without and with e.g. 5% of the organic solvent you are using and with plus minus 0.3 units of pH changed. By doing so you can compare results with ligands added with the set of acquired reference data to judge whether you have a real ligand induced effect of just a artifact coming from solvent or pH change. I guess a similar advice may also be helpful for non NMR readouts.

Kind regards

Alfred