I am using isothermal titration calorimetry to measure the binding affinity and stoichiometry between a small molecular weight poly(amino acid) (3500 - 4500 Da) (SYRINGE) and high molecular weight glycosaminoglycan (80,000 - 100,000 Da) (CELL). I have used the experimental design function on NanoAnalyze to optimise the syringe and cell concentration to yield a sigmoidal curve with an assumed stoichiometry of 10 (n=10). However, after running the experiment, I obtained the thermogram attached below. I have also attached the binding isotherm, which shows high standard deviation of the KD, delta H and n values, even after excluding the outliers. I have tried multiple experiments to optimise the concentrations and injection volumes. What can I do to improve this result?
The usual advice when obtaining poor-quality data such as this is to thoroughly clean the cell. Also, check that the syringe is not clogged and the injection motor is working properly.
Dear Saoirse Casey-Power
Can you show us a raw thermogram (without subtracting any baseline)? It seems that you may have used too short time intervals between injections.
Hi Adam B Shapiro
Thank you for your suggestions. I have tried to clean the cell thoroughly and replaced the titration needle but I am still obtaining similar thermograms.
Dear Saoirse Casey-Power
A slow process is probably occurring during titration (especially in the first half of the experiment). That causes the accumulation of the not-yet-bound molecules and changes the baseline. To overcome this, you should prolong the time intervals between injections. The signal should return to the baseline before the next injection. It may happen that you will have to use 5-10 times longer intervals (to be found experimentally).
Saoirse Casey-Power the ITC titration raw data (time vs heat rate) show poor quality - including Y axis baseline shifts between injections, as well as some heat changes going above the baseline and some below the baseline in an apparent random pattern. Have you checked the ITC results using a control experiment with no expected binding (like water titrated into water, buffer into buffer, or titrant injected into cell with buffer only no sample) that should give small, reproducible heat changes per injection.
How are you cleaning the ITC cell and syringe? You may need more rigorous cleaning methods. Check with the ITC supplier for suggested cleaning protocols.
How are you preparing the samples for ITC? What is the buffer?
Have you recently used the ITC for other samples that gave better-quality data? Do you have test samples recommended by your ITC supplier to test out performance?
Based on what you are showing, your issues could be instrument-related or sample related. You should consider contacting the the support team of the supplier of your ITC - they can suggest diagnostic tests to troubleshoot your ITC performance.
Hello Verna Frasca
I have carried out control experiments where the titrant was injected into the cell containing buffer alone. However, I noticed the same random changes in baseline, as attached below. The buffer is degassed, ultrapure water adjusted to pH 5.1 and this was used to prepare each sample. I have cleaned the ITC thoroughly using degassed ultrapure water, 0.5% (w/w) SDS detergent and a second flush of degassed ultrapure water, as per the NanoITC manual.
Water is not a buffer and does not provide buffering capacity. You can still have mismatched samples in the ITC cell and syringe. Do you check the pH after you prepare the samples?
Are other ITC users getting "good" data with the same instrument?
Does TA Instruments recommend a control titration like water titrated into water (with no binding), and/or a positive control experiment, that will test out the performance of the ITC.
If the control experiments pass, and other people are getting good data with the instrument, there may be an issue with your sample, like poor pH matching.
If several experiments fail to give useable data, You should consider there is an issue with the instrument. Your ITC cell and/or syringe could still be dirty, even with degassed samples a dirty cell can still result in bubbles. It may need more rigorous cleaning. Maybe there is an issue with injection and/or stirring. There are several possibilites.
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