I am using ITC to screen a library of DMSO-solubilized small molecules for binding activity to a drug target. For dealing with DMSO background contribution to injection heats, I know that the most common advice is to for one to add DMSO to their protein sample (the titrand in the cell) to make it the same DMSO concentration as what is in titrant sample in the syringe.
I was wondering is it equally effective to instead run a DMSO-buffer titration into buffer and subtract these injection heats from the DMSO-drug-buffer into protein-buffer titration injection heats? Is this a valid approach?
Also, are these two approaches equivalent? Please explain.
Thank you so much for your help!
When using ITC to screen small molecules dissolved in DMSO, the DMSO itself can contribute to the injection heats, potentially masking or interfering with the binding signal of the target molecule. To mitigate this effect, it's common to match the DMSO concentration in the protein sample (titrand) with that in the titrant sample.
Let us compare the two approaches:
Equivalence of the Two Approaches:
In general, matching DMSO concentrations is often considered the more reliable and straightforward approach. However, if you have reason to believe that the DMSO background signal is not linear or if you encounter significant noise in your data, the subtraction method may be worth considering
G. Plason Zuerkanah Plakar Hello, thank you for your help! I was wondering whether going the subtraction route might be susceptible to more noise. Your answer suggests that this is the case. Can you please clarify the last sentence? It seems like there was a typo. Did you mean to say that *matching DMSO concentrations* would be worth considering if the background signal is not linear or the data very noisy? Thanks again!
Gabriel Moreno, thank you for drawing my attention to the typo.
This is what I meant: In general, matching DMSO concentrations is often considered the more reliable and straightforward approach. However, if you have reason to believe that the DMSO background signal is not linear or if you encounter significant noise in your data, the subtraction method may be worth considering. Thanks.
Thank you so much for taking the time to help me. To clarify, I am confused because the indications (non-linearity and high noise) that you give in the concluding sentence of your answer in favor of considering the subtraction method over the matching method are the same ones given as contraindications for choosing the subtraction method in the preceding paragraph:
"Subtracting DMSO-Buffer Titration: [...] Accuracy: The subtraction process assumes that the DMSO-related background signal is consistent and linear throughout the titration. If there are any deviations from linearity, the subtraction may not be accurate. Noise: Subtracting two sets of data can introduce noise into the final result, potentially obscuring the binding signal."
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