The Road to Becoming a Calorimeter Master Vol.2 Continued
Let’s Use the Isothermal Titration Calorimeter iTC200!
* At the end of this article, you can download the materials.
Finally, Mr. Nakamura visited Dr. Fukada’s room. 
Dr. Fukada! It has been a long time!! How have you been?
Mr. Nakamura, welcome. I’ve been waiting for you. How long has it been since we last met? Hehe. It seems you are going to use ITC and DSC.
Yes, that’s right! Please guide me. I knew you were using it during my student days, but I never thought I would be the one using it.
Haha, the world works in mysterious ways. So, where shall we start?
I was measuring using the iTC at the company, but it wasn’t going well… so, I compiled some questions.
Wow, you’re well prepared. Let’s take a look.
Dr. Fukada examined Mr. Nakamura’s notes.
Mr. Nakamura. Have you not used the system for a while?
High DP and increased baseline noise level are signs.
Switching the reference cell water did not improve it, right?
I think you should thoroughly clean it first.
Also, cleaning remains crucial in the future.
In my case, I clean the cells and syringes with detergent and ultrapure water after each measurement.
Then, the high DP over the reference Powder was due to dirt in the sample cell, not air bubbles, right?
I believe so. Clean it while warming the cells with 20% Contrad 70 or 14% Decon 90. It should be in the manual.
Oh, here it is! (Manual P41 and Note 1)
(Note 1) For VP-ITC users, please refer to manual P.45. To change jacket temperature, input the temperature in Set Point (℃) in the Thermstat/Calib. tab and click Set Jacket Temp.
Teacher, if DP is low, I suspect air bubbles are in the sample cell. How can we prevent air bubbles in the cell?
When injecting the solution, take a little more solution (350 μL) in the syringe and insert it slowly, keeping the needle as close to the bottom of the cell as possible. Usually, this should suffice, but for minute amounts, like around 50 μL, pump to create convection inside the cell to eject air bubbles.
The tube volume to the cell is roughly 50 μL, so if you pump more than that, you’ll reintroduce bubbles after taking them out of the cell. Be cautious about that.
Also, gently shake the syringe up and down inside the cell to help remove bubbles that tend to linger at the boundary between the cell and the conduit.
Additionally, this is important! If the solution’s temperature is lower than the measurement temperature, bring it back to a temperature close to the measurement temperature. According to the manufacturer, degassing the sample is generally not required (Note 2) for iTC200.
As you repeat the measurements, you’ll get the hang of it, so keep at it patiently.
(Note 2) For VP-ITC users, please degas before measurement.
About the reference, why is ultrapure water not a problem? Usually, in experiments, isn’t it better to match the buffer?
The reference cell needs to cancel out external temperature fluctuations by filling it with a solution with the same heat capacity as the sample cell. Water has high heat capacity, and wouldn’t water be the most content-rich in the solution? That’s why it’s okay to use water instead of a buffer for the reference cell.
If you replace the reference water once a week, it should be fine, but since it evaporates slightly, replacing it each time during high-temperature measurements may be necessary.
I see, that’s how it is.
Teacher, may I ask one last thing?
Feel free to ask more than one.
Thank you! Regarding the concentration settings for actual sample measurements, the manual mentions the C value as a standard, but I can’t seem to get the concept.
Let me add. The C value is a guideline for determining the cell sample concentration necessary to produce a good sigmoidal curve during analysis.
Since the iTC200 comes with simulation software in the control software, you can easily set conditions using that.However, generally speaking, a protein concentration of about 10 μM is often used, but it fluctuates according to the binding strength, so be careful.
Setting the concentration based on the C value results in a very low cell concentration in cases of high-affinity interactions.
For instance, for a sample with a dissociation constant of 10 nM, to fit a C value of 5,
you can calculate that 50 nM is the cell concentration.
But, when you actually measure at that concentration, there might be insufficient heat, so it’s preferable to ensure at least 1 μM (Note 3).
Accurate concentration information is very important for ITC measurements, but we will discuss it another time. For now, determining the protein concentration from the UV absorption spectrum around 280 nm is a good approach.
Let’s try measuring it for real. For practice, let’s use CaCl2 and EDTA at first.
(Note 3) The heat change obtained by interaction varies. Sometimes 1 μM is sufficient, but if the change is small, you need to increase the cell concentration for measurement.
Next time, we will look at the actual obtained data, learn key points from the data, and tackle trouble handling methods. Stay tuned!
When writing this blog, to make it more realistic, I visited Dr. Fukada while he was conducting an ITC measurement.
The system he was using was the OMEGA, which was launched in 1987. I was truly surprised to see a near 30-year-old system operating perfectly. It was my first time actually seeing it in operation. The PC being used was running Windows 3.1!? No USB ports, of course. But the most surprising thing was the cell in the system had never malfunctioned, with only the stirrer motor and PC board requiring repairs.
Dr. Fukada understood the system’s nature and developed his own unique usage method not written in the manual, emphasizing thorough cleaning after measurements.
Mixing the knowledge gained from Dr. Fukada, I will continue to share information with everyone in the future.
The history of MicroCal ITC can be found on the blog of Malvern’s UK headquarters. Check out the material talks here!
