I am trying to measure the binding between an aptamer (ssDNA) that 'supposedly' binds specifically to (+)-methamphetamine. The aptamer comes as a solid form, which I initially dilute in H20, and then dilute to my desired concentration (20uM) in a buffer. Methamphetamine comes in a 1mg/mL solution (in methanol) that I dilute to its desired concentration (200 uM) in the same buffer as above.
The attached graphs show the data of
(A) buffer to buffer,
(B) buffer + meth (200uM), and
(C) aptamer (20 uM) + meth (200 uM).
Why am I getting such high heats of dilution for the aptamer + meth data? How do I interpret this? Does this show binding?
There is a huge exothermic heat response in each titration thermogram of the methanol-dissolving (+)-methamphetamine titration to Buffer (panel B).
It is due to the dilution heat of methanol like when you try to make a diluted methanol solution with water in a glass beaker. The beaker holding the diluted methanol will turn much hotter than before.
Such huge exothermic heat background will,
1) surpass the dynamic range of ITC differential power.
2) shadow the exothermic heat of binding events, If the heat response is way smaller than the dilution heat.
You can subtract the isotherm of the panel B from panel C to get the “net” isotherm, which is attributed to extra heat from DNA aptamer binding to (+)-methamphetamine.
I am aware of that the number of Y axis of panel C is extremely big and inconsistent to panel B. You need to double check if you provide a correct concentration of the DNA aptamer for the ITC analysis.
Best of luck,
Yun-Tzai
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning