Isothermal Titration Calorimetry (ITC) measures heat of interaction between two molecules. In ITC a syringe containing a "ligand" is titrated into a cell containing a solution of the "macromolecule". As the two elements interact, heat is released or absorbed. When the macromolecule in the cell becomes saturated with added ligand, the heat signal diminishes until only the background heat of dilution is observed. An example of ITC data collected in the Biophysical Resource. A good review for ITC application for protein studies is given by Pierce et al. The Biophysical Resource at Keck Facility offers the VP-ITC from MicroCal as an "open access" instrument that can be used by trained users.
To schedule a training session please contact Ewa Folta-Stogniew
For ITC schedule go to Scheduler
ITC performs the measurement on native molecules in solution; no modifications are needed and the measurement does not require any consumable.
|VP ITC from Microcal:|
Ligand Syringe Volume:
Single titration time:
|30 - 50 minutes (fully automated)|
|5 - 15 minutes|
|either with the data station of the computer, or the software can be installed in the individual research laboratories for data analysis|
General guideline for sample requirements can be printed from “Sample requirements” (as suggested by MicroCal- manufacturer of the ITC instrument).
Detailed sample preparation guideline is given in "ITC_sample_preparation". The estimation for protein and ligand concentration needed for ITC experiment can be made following the suggestions discussed in “Experimental Consideration_ITC”.
The ITC instrument in Biophysical Resource has a 250 uL titration syringe. The MINIMAL requirements can be summarized (FOR A SINGLE TITRATION):
|2.1 mL of 3 uM (for tight binding i.e. binding where Kd is smaller than 1 uM); otherwise 15 uM (or higher)|
|700 uL at concentration 15-20 times higher than the concentration of macromolecule|
|20 mL of sample's buffer (for cell rinsing between titrations)|
(if possible higher conc. should be used to assure a better signal)
ITC is an universal tool for monitoring binding reactions because it measures binding from the universal heat/enthalpy change associated with all binding interactions. The analysis is performed at equilibrium, in solution phase and without any labeling or need for a fluorescent or other probe. An ITC experiment provides direct information about the thermodynamics of binding. The enthalpy of binding is measured directly and when combined with the equilibrium binding constant (also measured directly) yields the entropy change. The temperature dependence of the enthalpy changes yields the heat capacity which provides insight into the surface area buried during binding. ITC can also be used to determine the mass ratio between interacting species; however, the absolute stoichiometry of the final complex needs to be verified by other methods.
To schedule a training session please contact: Ewa Folta-Stogniew
First time users of the resource follow the booking and charging protocol; please fill out the Biophysics Resource Usage Form linked in the box on the left, and email the completely filled usage form, with email Subject: "Biophysics Resource Usage," to Ewa Folta-Stogniew.
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