Leeds is one of the few Universities in the UK equipped with two automated SPR devices, a BIACORE 2000 (Biacore International SA) in the laboratory of Prof P.G. Stockley, and BIACORE 3000 in the Wellcome Trust Centre for Biomolecular Interactions.
SPR - Surface plasmon resonance is a phenomenon which occurs when light is reflected off thin metal films. A fraction of the light energy incident at a sharply defined angle can interact with the delocalised electrons in the metal film (plasmon) thus reducing the reflected light intensity. The precise angle of incidence at which this occurs is determined by a number of factors, but in the Pharmacia BIA devices the principal determinant becomes the refractive index close to the backside of the metal film, to which target molecules are immobilised and addressed by ligands in a mobile phase running along a flow cell. If binding occurs to the immobilised target the local refractive index changes, leading to a change in SPR angle, which can be monitored in real-time by detecting changes in the intensity of the reflected light, producing a sensorgram. The rates of change of the SPR signal can be analysed to yield apparent rate constants for the association and dissociation phases of the reaction. The ratio of these values gives the apparent equilibrium constant (affinity). The size of the change in SPR signal is directly proportional to the mass being immobilised and can thus be interpreted crudely in terms of the stoichiometry of the interaction. Signals are easily obtained from sub-microgram quantities of material. Since the SPR signal depends only on binding to the immobilised template, it is also possible to study binding events from molecules in extracts; i.e. it is not necessary to have highly purified components.
Surface plasmon resonance detection unit. L: light source, D: photodiode array, P: prism, S: sensor surface, F: flow cell. The two dark lines in the reflected beam projected on to the detector symbolise the light intensity drop following the resonance phenomenon at time = t1 and t2. The line projected at t1 corresponds to the situation before binding of antigens to the antibodies on the surface and t2 is the position of resonance after binding.
The BIACORE 2000 and the BIACORE 3000 instruments use the same sensor chip technology: a thin gold film on a glass slide divided into four channels, or flow cells, to which ligands can be immobilised by several methods including amine, nickel-histidine or streptavidin-biotin coupling.
Both instruments can record from all four flow cells simultaneously, allowing real-time reference subtraction and measurement of analyte binding to 3 different ligands. The Biacore3000 has better signal to noise characteristics than the 2000 device, making it suitable for measuring binding of small analytes (as small as 180 Da), while the 2000 device has versatile sample recovery functions enabling downstream processing of material which has bound to the ligand.
The raw data are presented as a real-time graph of response units (RU) against time. This graph is referred to as a sensorgram. (For proteins 1000 RU represents about 1 ng bound to the flow cell surface.) During injection of analyte changes in signal result from two processes: association to and dissociation from the surface. At the end of injection, running buffer continues to flow over the chip; at this stage the change in signal results from dissociation only.
Significant conclusions can be drawn from the initial sensorgram. E.g., is there any interaction? Which analyte binds the strongest? Do the regeneration conditions return the signal to the initial baseline? The results can be further analysed using the program Biaeval 3.0 which facilitates display of multiple cycles of analysis in one plot window and the kinetic evaluation of data. Several different mathematical models are implemented, and further models can be constructed.
Plots can be copied to the clipboard and pasted into Windows applications such as Word. The raw data can be exported as lists of numbers for use in other analysis or plotting programs. Users are expected to analyse and archive their own data.
Because of the very narrow liquid path formed by the microfluidic system, all buffers must be filtered through a 0.2 micron filter, and de-gassed before use, and must contain 0.005% (w/v) surfactant P20 to reduce fouling of the inner surfaces. Regular cleaning routines are carried out to prevent build up of adsorbed protein and growth of bacteria in the liquid handling system.
Samples for analysis (e.g. proteins) should be dialysed or de-salted in the running buffer before dilutions are made, to avoid large changes in signal due to refractive index differences. Typically an experiment will consist of 6 to 10 injections of 0.1 to 0.2 ml of very dilute analyte, each injection being followed by an injection of regeneration solution to displace remaining analyte. 200 - 500 ml of running buffer will be consumed.
The SPR facility is the responsibility of Iain Manfield. Both instruments are operated as a University facility and need to be maintained regularly by the manufacturer. An access charge is levied per day in order to recoup these costs, and in addition users have to provide their own consumables. Users must be given training before operating the machines. This, and expert assistance, can be provided by the Facility Manager, Iain Manfield (telephone: 0113 343 7279, email: firstname.lastname@example.org).
Booking instrument time
A booking diary is maintained by Iain Manfield. To arrange time slots contact him, either by email or personally in Room 9.108h, Astbury Building. Three working days notice must be given for cancellation, otherwise the time will still be charged for.
"Probing the molecular mechanism of action of co-repressor in the E.coli methionine repressor-operator complex using surface plasmon resonance". (1995) Nucleic Acids Res. 23, 211-216. I.D.Parsons, B. Persson, A. Mekhalfia, G.M.Blackburn and P.G.Stockley.
"Quantitation of the E. coli methionine repressor-operator interaction by surface plasmon resonance is not affected by the presence of a dextran matrix." (1997) Anal. Biochem. 254(1), 82-87. Isobel D. Parsons and Peter G. Stockley
"Dissecting the molecular details of prokaryotic transcriptional control by surface plasmon resonance: the methionine and arginine repressor proteins." (1998) Biosensors. Bioelect. 13, 637-650. Peter G. Stockley, Andrew J. Baron, Catherine M. Wild, Isobel D. Parsons, Coleen M. Miller, Carol A. M. Holtham and Simon Baumberg.
"A biaryl peptide crosslink in a MetJ peptide model confers cooperative nonspecific binding to DNA that ablates both repressor binding and in vitro transcription.” (2003) Bioorganic & Medicinal Chemistry 11(6), 811-816. J.C. Yoburn, S. Deb, I.W. Manfield, P.G.Stockley and D.L. Van Vranken.
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