Equipment available:PEAQ ITC
Equipment location: New Biochemistry 00-059
Equipment coordinator: Dr David Staunton
Equipment charge: £150/day
Isothermal titration calorimetry
Calorimetry is the measurement of heat evolved or absorbed during a chemical or physical change in a sample. Isothermal titration calorimetry is conducted at a constant temperature with small aliquots of one reactant ("ligand") are titrated into a larger volume of a second component ("sample") in solution, and the resulting heat evolution/absorption is measured. The area under the heat evolution curve for each aliquot is measured and then replotted as a function of the molar ratio of ligand to sample. The resulting titration curves can be analysed to give the enthalpy ΔH of the interaction, and the association constant K, which is related to free energy ΔG; from these the entropy ΔS can be calculated. These are absolute thermodynamic measurements.
- · Isothermal titration calorimetry (ITC) is mostly used to measure strengths of interaction between proteins and ligands: either small molecules like drugs, or large ones such as nucleic acids or other proteins.
- · It can also be used to follow the dissociation of a complex as it is injected into buffer.
Malvern PEAQ-ITC microcalorimeter
This instrument uses the smaller sample and reference cells of the older Microcal iTC200 but has much greater signal sensitivity. The Malvern software converts the heat evolution traces to ΔH values and can analyse for single site binding and some complex models of interaction. It also has an experiment modelling software that allows the users to refine their experimental design. Other freeware that can analyse the raw data is NITPIK from the NIH group of Peter Schuck.
The minimum sample volume for the cell is 0.25ml and for loading the syringe 70µl is required (although only 40µl is actually used in the syringe).
It is strongly recommended that ITC be used to confirm a KD value rather than used to scout for it as the approximate KD is required for successful experimental design. The following conditions can be used detect KDs of 10 µM to 10 nM and is ideal for KDs of 2 µM to 100 nM.
· 100µM Ligand in the Syringe
· 10µM Sample in the cell
· 19 x 2µl injections
Any chemical potential difference between the ligand and sample will generate heat, and therefore signal, when mixed and so their buffers must be identical. The best approach is to dialyse both ligand and sample against the same buffer before the experiment but the samples should not be concentrated afterwards as the wetting and preservative agents on the concentrator membranes will introduce chemical potential differences.
A heat of dilution experiment should normally be conducted where the ligand is injected into buffer only to observe any signals due to these buffer differences which can then be removed from the experimental data. Even dialysed samples will not have exactly identical buffer composition due to the macromolecules affecting the ion distribution across the dialysis membrane.
Common biological buffers such as TRIS and HEPES are not recommended for ITC due to their high heats of ionisation and phosphate buffered saline is normally used.
Avoid DTT as a reducing agent as it is unstable, undergoes oxidation and has a high background heat. beta-mercaptoethanol and TCEP are better for ITC but TCEP is not stable in phosphate buffer.
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