Introduction

DSC

ITC

PPC

Instruments

Sample preparation

Buffers

User Notes

Accomodation

How to find us

Costs

Literature

Suppliers

Homepage

Glasgow Biophysical Chemistry Group Biological Chemistry Section Please note: Alan Cooper is now formally retired (as of September 2010) and we are currently unable to provide microcalorimetry facilities to external users. However, we will retain this website so that colleagues may still access useful information and user notes. The Glasgow Biological Microcalorimetry Facility was funded jointly by BBSRC and EPSRC from 1989 to 2007, with up to 40% of instrument time available on an informal basis for members of the academic community and others involved in biomolecular research and development. Professor Alan Cooper Chemistry Department, Joseph Black Building University of Glasgow, Glasgow G12 8QQ Tel: (+44) (0)141-330 5278 Fax: (+44) (0)141-330 2910 email: alanc@chem.gla.ac.uk

• VP-DSC
• PPC (accessory to VP-DSC)
• VP-ITC

For ITC experiments, both macromolecule and ligand must be in identical buffer/solvent otherwise large heats of dilution will mask the desired observation. This is best achieved by exhaustive dialysis of the macromolecule against appropriate buffer, using the final dialysis buffer to make up the ligand solution. If both components are macromolecules, they may both be dialysed in the same pot. Again, adequate volumes (50+ ml) of the actual dialysis buffer should be supplied for instrument equilibration and baseline controls. Typical Concentrations: For the macromolecule (in the ITC cell) concentrations should normally be at least 10 micromolar (i.e. 0.25 mg/ml for a 25kDa protein). The ligand solution (in the injection syringe) should be 15-20x higher in molar concentration, or even higher for weaker binding systems. (This is to take account of dilution of the ligand during the experiment. At the end of the titration, typically 250ul of ligand will have been added to 2ml of macromolecule.) Sample volumes: at least 2ml of "macromolecule" and 0.4ml of "ligand" per titration. More is better, especially on untried systems where additional control experiments may be necessary.

Truly quantitative data can only be obtained if molar concentrations of proteins/macromolecules and ligands are known accurately. This can usually be done on-site by UV/vis absorbance measurements, provided molar extinction coefficients are available. Please contact us for advice on sample handling/measurement if you have any doubts/queries.

PPC requires quite high concentrations (2-5 mg/ml) and is not advised except for specialist applications on well-characterized systems.

• Sturtevant (1987) Ann.Rev.Phys.Chem. 38, 463-488.
• Privalov (1980) Pure & Appl.Chem. 52, 479-497.
• Cooper & McAuley-Hecht (1993) Phil.Trans.Roy.Soc.Lond.A 345, 23-35.
• Cooper & Johnson (1994) Meth.Mol.Biol. 22, 125-150.
• Cooper (1997). Microcalorimetry of protein-protein interactions. Methods in Molecular Biology Vol.88: Protein Targeting Protocols, pp.11-22 (ed. Roger A. Clegg, Humana Press 1997).
• Cooper (1998) Microcalorimetry of protein-protein interations, in "Biocalorimetry: the applications of calorimetry in the biological sciences", eds. J.E.Ladbury & B.Z.Chowdhry (Wiley), pp. 103-111.
• Cooper (2000) Microcalorimetry of protein-DNA interactions, in "Protein-DNA Interactions: A Practical Approach", ed. A.Travers & M.Buckle (Oxford Univ.Press)pp.125-139.
• Cooper (1999) Thermodynamics of protein folding and stability. "Protein: A Comprehensive Treatise", Volume 2, pp. 217-270. (Editor: Geoffrey Allen. JAI Press Inc., Stamford CT, 1999)
• Cooper (1999) Thermodynamic analysis of biomolecular interactions. Curr.Opinion Chem.Biol. 3, 557-563.
• A. Cooper, M. A. Nutley, A. Wadood, Differential scanning microcalorimetry in S. E. Harding and B. Z. Chowdhry (Eds.), Protein-Ligand Interactions: hydrodynamics and calorimetry. Oxford University Press, Oxford New York, (2000) p 287-318.