BBSRC/EPSRC Biological Microcalorimetry Facility

Professor Alan Cooper
Chemistry Department, Joseph Black Building
University of Glasgow, Glasgow G12 8QQ

Tel: (+44) 141-330 5278
Fax: (+44) 141-330 2910
email: alanc@chem.gla.ac.uk

The Glasgow BBSRC/EPSRC Biological Microcalorimetry Facility is an informal service available for external users - at minimal cost* and administratively uncomplicated for academic researchers. Enquiries welcome.

Contact: Alan Cooper (email: alanc@chem.gla.ac.uk) or Margaret Nutley (email: margaret@chem.gla.ac.uk)

Samples may be sent to Glasgow for analysis by us, or visitors welcome (see below for recommended accommodation.) Training is provided for new users. Service includes full analysis/interpretation (using Microcal ORIGIN and related software) and preparation of publication-ready figures.

*Starting January 2000, the research councils require that we introduce a graduated scale of (modest) charges for academic users. However, genuine inability to pay will not disqualify legitimate users! See below for details.

A range of instruments is currently available:-

***Stop Press: NEW Pressure Perturbation Accessory now installed (August 2000).

For more details on the instrumentation, contact MicroCal Inc. or their UK/European agents (Heath Scientific).

Microcal MCS and VP-DSC:

Differential scanning calorimeters (DSC) for the study of thermal transitions in dilute solution. (Protein folding, DNA melting, lipid bilayer transitions, etc.) Typical protein concentrations 0.1-3 mg/ml, 0.7 - 1.8ml sample volume, temperature range 5 to 130oC, depending on instrument. Isothermal and down-scan modes are also available (VP-DSC).

Microcal OMEGA, MCS and VP-ITC:

Isothermal titration microcalorimeters (ITC) for binding studies in dilute solution (enzyme-ligand, protein-protein, protein-nucleic acid, and/or small molecule interactions). Typical (macromolecule) concentrations down to 10 micromolar in the reaction cell (2ml volume), with 15-20x higher concentrations of titrant (ligand) in the injection syringe (min. 400 microlitre required). Temperature usually 25oC, but 2-50oC range possible. Dissociation/dilution methods are also available.

Sample Preparation:-

Samples for DSC should be properly equilibrated in an appropriate buffer - usually by dialysis - and adequate volumes (50+ ml) of the actual dialysis buffer should be supplied for instrument equilibration and baseline controls. Typical sample concentrations: 0.1 - 3 mg/ml, depending on system and quality of data required. Sample volumes: at least 0.7ml (VP-DSC) or 1.8ml (MCS) per experiment. More is better.

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.

Buffers:-

Most buffers are compatible with ITC/DSC cells, and detergents and/or organic solvents may be incorporated as well - provided appropriate precautions are taken to minimize heat of dilution artefacts, etc. Bear in mind that, at the relatively high ligand concentrations sometimes needed for ITC, the buffer capacity may not be adequate, and some pH adjustment may be necessary. It is best to choose a reasonably high buffer concentration (e.g. 0.1 M) if your system can take it. Please avoid mercaptoethanol in your buffers - it smells nasty and, more important, can cause erratic baseline drift in the microcalorimeters. DTT or other reducing agents are acceptable, if required by your sample.

Pressure Perturbation Accessory:-

This is a new development from Microcal that allows determination of volumetric properties of proteins and other macromolecules in solution over a wide range of temperatures. We are still evaluating the system. Sample requirements are as for VP-DSC, but somewhat higher concentrations are needed (2.5 mg/ml or higher). Watch this space.

Background references:-

Click here for examples of sample publications.

Reservations...

Requests for ITC/DSC use should be made a reasonable time in advance, either by phone or email to Alan Cooper or Margaret Nutley at the addresses given above. Currently the schedule is typically booked up 3-4 weeks in advance (sometimes longer in busy periods) - but please contact us if you have urgent requests.

Accommodation

We recommend the Amadeus Guesthouse or Heritage Hotel, both of which are within reasonable walking distance of the University and adjacent to the underground station for easy access to the city centre. Click here for other nearby accommodation or here for more general information about Glasgow. See also campus maps and how to get here. University/campus accommodation is sometimes available - please contact the University Accommodation Office.

Microcalorimetry Suppliers/Manufacturers...

What the service costs...

Inability to pay is no bar to legitimate academic users, but we do advise you to include provision for such costs in grant applications. But, for those who can afford it, the minimum daily rates for academic users* agreed with BBSRC/EPSRC as a contribution to consumable and maintenance costs, are as follows:-

As a rough guide, one working day is sufficient for 2-3 ITC titrations or DSC scans, with appropriate controls.

*Rates for non-academic/commercial users are much more realistic(!), subject to negotiation with the University of Glasgow Research and Enterprise.


Return to Alan Cooper home page.