Isothermal Titration Calorimetry Assay Services

ITC is a solution based assay that measures the heat released or absorbed as the result of a chemical reaction.

During the assay, one component of the reaction is contained in a temperature-controlled cell, then the binding partner is gradually titrated into the system. When binding occurs, heat is either released or absorbed. This heat change is directly proportional to the amount of binding. This technique is suitable as a secondary screen yielding not only the binding affinity but also the thermodynamic profile of a ligand-target interaction giving insight into the structure-function relationship on the molecular level.  

  • Label-free technique avoiding any influences of labels or immobilization on the binding reaction
  • Deliverables: binding stoichiometry, binding enthalpy (∆H), entropy (∆S), binding affinity KD

ITC Principle

A solution with the agent is gradually titrated via an injection syringe into a solution with target molecules in a stable temperature environment. The heat absorbed or generated during the binding reaction is measured and allows the calculation of various interaction parameters.

isothermal titration calorimetry principle

Assay Details

Thermodynamic profile

isothermal titration calorimetry example curve

The heat released or absorbed during titration of the ligand into the target solution is directly proportional to the ligand-target binding which provides a thermodynamic profile of the molecular interaction.

The integrated area of each injection peak is plotted against the molar ratio of ligand to target. The isotherm is fitted to a binding model in order to derive binding affinity (KD) and reaction stoichiometry (n).

Example: BRD4

isothermal titration calorimetry example for compound binding to a target

Example of evaluation of BRD4 bromodomain 1 (BRD4-1) binding to JQ1

Upper image: Overlay of titration spectrograms of BRD4-1 protein vs. JQ1 (black) and buffer (blue).

Lower image: integrated data for BRD4-1/JQ1 binding was analyzed using one-site binding model. The best fit parameters are shown in the box. JQ1 shows binding to BRD4-1 with an estimated KD of 36 nM.

Screening Details

Instruments: The low volume nano ITC (TA instruments) is highly sensitive and uses less sample than the standard volume ITC instrument. The cell is constructed of inert gold with high thermal conductivity. Cylindrical cell geometry maximizes stirring efficiency and eliminates dead zones and bubbles.

Turnaround time: The study is typically completed within 2 weeks after receipt of materials. Projects are performed on a first-come-first-serve basis.

Sample requirements: Protein purity of 90% or more is required. The protein amount is project-dependent. Typically, 1-5 mg of pure protein or more will be needed.
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Screening location: The assay is performed in Malvern, PA, USA.

 

Applications of Isothermal Titration Calorimetry

ITC services

Direct binding

  • Ligand into protein vs protein into ligand
  • Reverse titrations for poorly soluble compounds
  • Main limitation is maximum protein concentration

Competitive replacement

  • Necessary for compounds with KD<1 nM
  • Weaker compound is required

Enthalpy screening

  • Determine binding enthalpy
  • Higher throughput
  • Reduced protein usage