Nano ITC (Isothermal Titration Calorimeter) directly measures heat evolved or absorbed in liquid samples as a result of mixing precise amounts of reactants. A spinning syringe is utilized for injecting and subsequent mixing of reactants. Spin rates are user selectable. It is also the only method capable of determining the enthalpy, entropy, and the Gibbs free energy of a reaction in a single titration experiment. Temperature differences between the reference cell and the sample cell are measured, calibrated to power units and displayed to the user as well as saved to disk. The data channel is referred to as the DP signal, or the differential power between the refrence cell and the sample cell. An injection which results in the evolution of heat (exothermic) within the sample cell causes a negative change in the DP power since the heat evolved chemically provides heat that the DP feedback is no longer required to provide. The opposite is true for endothermic reactions. Since the DP has units of power, the time integral the peak yields a measurement of thermal energy, ΔH. This heat is released or absorbed in direct proportion to the amount of binding that occurs. When the macromolecules in the cell becomes saturated with added ligand, the heat signal diminishes until only the background heat of dilution is observed. With the Nano ITC system the entire experiment takes place under computer control. The user inputs the experimental parameters (temperature, number of injections, injection volumes) and the computer carries out the experiment. Origin software is then used to analyze the Nano ITC data using fitting models to calculate reaction stoichiometry (n), binding constant (Kh), enthaply (ΔH) and enthropy (ΔS).
Minimal requirements can be summerized for a single titration (if possible higher concentration should be used to assure a better signal). The syringe containing "ligand" is titrated (injected) into a cell containing a solution of a "macromolecule": Macromolecule solution: 2.1 mL of 3 µM (for tight binding i.e. binding where Kd is smaller than 1 µM); otherwise 15 µM (or higher). Titrant: 700 µM at concentration 10 times higher than the concentration of macromolecule. Additional buffer: 20 mL.
Equipment
Instrument: Nano ITC Microcalorimeter
Cell: Nano ITC gold cylidrical cell (1 mL)
Degassing Station: stirring platform, vacuum chamber, vacuum port, temperature control and electronic timer.
Temperature: 25oC - 80oC
Titration Syringes: 250 µL
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