How does isothermal titration calorimetry?
Isothermal Titration Calorimetry (ITC) is a technique used in quantitative studies of a wide variety of biomolecular interactions. It works by directly measuring the heat that is either released or absorbed during a biomolecular binding event.
How sensitive is ITC?
The MicroCal PEAQ-ITC is a highly sensitive, low volume isothermal titration calorimeter for the label-free in solution study of biomolecular interactions. It delivers direct measurement of all binding parameters in a single experiment and can analyze weak to high affinity binders, using as little as 10µg sample.
What is the ITC protocol?
Protocol. Isothermal titration calorimetry (ITC) is a well established technique that can determine all the thermodynamic parameters (affinity, enthalpy and stoichiometry) of a binding interaction in one experiment. 1. ITC works by titrating one reactant into a second reactant under isothermal conditions.
What is the C value in ITC?
The c-window is described by equation 4, where K is the binding constant and n is the number of ligands that will bind the macromolecule: c = nKa[titrand] or c = n[titrand]/Kd An acceptable range for the c window is 1< c < 1000, with ideal values falling between 5 and 500 (2).
Is an isothermal process?
An isothermal process is a thermodynamic process in which the temperature of a system remains constant. The transfer of heat into or out of the system happens so slowly that thermal equilibrium is maintained.
How accurate is ITC?
Summary. ITC provides the most accurate measurements of binding affinity and has become the gold standard to gauge the accuracy of other techniques. In addition, ITC is also the only technique capable of measuring the thermodynamic components of the binding energy, the enthalpy and entropy changes.
What is molar ratio in ITC?
Indian Institute of Technology Delhi. N is a measure of the binding ratio as much as the activity of one’s samples. The X-axis value (Molar Ratio) at the mid (inflection) point of an ITC sigmoidal curve can be conveniently seen as the stiochiometric coefficient (N-value).
How much protein is typically required for an ITC experiment?
No immobilization required → measurements in solution. Low sample consumption for ITC standards → only 300 µl of a 20 µM protein solution required.
What affects binding affinity?
Binding affinity is influenced by non-covalent intermolecular interactions such as hydrogen bonding, electrostatic interactions, hydrophobic and Van der Waals forces between the two molecules. In addition, binding affinity between a ligand and its target molecule may be affected by the presence of other molecules.
Why is internal energy 0 for isothermal?
In an isothermal process, temperature of the system remains constant. Thus, the internal energy of the system also remains constant. Hence the change in internal energy is 0.
How much protein do you need for ITC?
Typical protein concentrations for ITC are 10 to 50 uM with a 1.8 ml sample volume which is placed in the cell. The ligand (protein, small molecule, nucleic acid) concentration is typically 50 to 500 uM with a volume of around 500 ul for the syringe. In general the ligand concentration is 10X higher than the protein.