Hello, I'm Sanam , a grad student new to ITC. I am trying to do titration between a protein and small molecules that we knew it binds to the protein since it's a commercial inhibitor. I purified target protein . For doing titration the protein concentration was 0.0501 mM( determined by uv absorbance at 280 nm and extension coefficient), and compound concentration was 0.59 mM. However, I got that result (attached file). I will really appreciate if someone could guide me a d help me. I am so new in ITC. By the way, I used TA affinity ITC. Thanks in advance.
Dear Sanam Mohammadzadeh
The first thing you can try is to analyze the data you show us. You should set up in NanoAnalyze a shorter time for integration of the heat after each injection (probably about 100 seconds). Then, in the "Modeling" tab, uncheck the first and probably the fourth injection. Then, show us a result, and we can try to suggest the next step.
Regards
Tomasz
Dear Dr. @Tomasz Fraczyk,
Hi, thank you so much for the reply. The n value should be 1 and the model should be independent. Please find the attached.
Dear Sanam Mohammadzadeh
Several issues can impair the signal in your experiment:
1. Too low signal-to-noise ratio. If this is your case (it could be), the first thing I suggest is repeating the experiment with 10 injections of 4 uL volume and increasing concentrations of protein/inhibitor at least two times. These steps will increase the signal/noise around four times.
2. At a specific temperature, you can have zero enthalpy change for the binding. Then, I would do a titration at a different temperature (at least 10 degrees lower or higher).
3. It can happen that the binding reaction is associated with protonation/deprotonation of the protein or the interacting molecule. Then, buffer molecules deprotonate/protonate. Thus it may appear that the heat of deprotonation/protonation of the buffer cancels out the heat of binding. If so, changing the buffer can help.
Do you use low-volume (190 uL) or standard-volume (950 uL) cell in your TA ITC? Based on injection volumes, I assume you have a low-volume cell, but I want to clarify it for further discussion.
I hope it helps.
Regards
Dear Dr. Tomasz Fraczyk ,
I really appreciate your reply. I am going to repeat the experiment and If you do not mind, I will send the results of titration to you. And yes, I do have a low- volume cell. Thank you very much for your helps and suggestion.
With the best wishes,
Sanam.
Dear Sanam Mohammadzadeh
You are welcome. I will gladly help and keep my fingers crossed for your next experiment.
Best regards,
Tomasz
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