The ISMB Biophysics Centre
at Birkbeck

Stopped-Flow
(KinetAsysT, Hi-Tech Scientific)


Our KinetAsysT can measure UV/Vis absorption, single or dual-channel fluorescence with a dead time of 1 to 2 milliseconds. Many enzymes, other macromolecules, substrates or products are optically active or fluorescent. Using this to measure rapid reaction kinetics can be critical to understand enzyme mechanism, ligand binding or the folding or assembly of complexes. The dual-channel setup allows FRET measurement and therefore distance measurements between fluorescent labels before and after a conformational change.

Various size syringes are available to allow mixing at different ratios. Double-mixing of samples is also possible to further investigate reactions of more than one step. The syringes and optical cell are immersed in a thermostatted waterbath allowing choice of reaction temperature.



Quench-Flow
(RQF-3, Kintek)


A quench-flow apparatus enables rapid stopping of a reaction by adding a chemical quenching agent to the reaction mixture. The shortest time points accessible lie in the range of milliseconds, so even the fastest reaction rates become accessible. The quench-flow is especially used for establishing single-turnover rates of enzyme-catalysed reactions when no optical probe is available and for isolating intermediate species.

The RQF-3 requires as little as 15 microlitres per timepoint. It produces the quenched samples that will then have to be analysed by an independent method. For example, there is a HPLC at the centre which may be used to determine the quantities of substrate and products in those samples.



UV/Vis Spectrometer
(Cary100, Varian)


For initial enzyme characterisation classical turnover experiments are very important. These give direct information about the catalytic rate of a reaction as well as on substrate binding and specificity. Also, enzyme inhibitors can be characterised easily.

The Cary100 is a dual beam instrument ideal for kinetic measurements as fluctuations in lamp intensity to not distort time courses. It has an 8-position sample changer where every sample has its own reference. Comprehensive software allows easy setting up of kinetic scans.

For measurement of slow fluorescence time courses, please refer to our fluorimeter page.






 
ISMB Biophysics Centre, Birkbeck, University of London
Last modified 2011