Fluorescence Polarisation Technique
The fluorescence polarisation technique can be used with stopped-flow instruments to allow anisotropy measurements as well as total fluorescence measurements to be made. This is achieved by simultaneously detecting the fluorescence contributions of a sample from light polarised perpendicular and parallel to the parallel plane of the excitation beam. Using a fluorescence polarisation accessory, a near collimated excitation beam is delivered to the sample cell through a single fibre optic system.
This arrangement allows the use of a calcite prism polariser with transparency down to 220 nm. The sample cell is configured in a T-format for dual channel fluorescence detection and the photomultiplier couplings are fitted with UV dichroic sheet polarisers. The polarisers are arranged to pass light in the parallel and perpendicular planes with respect to the parallel plane of the excitation beam. The signals from each photomultiplier are processed and displayed using either HI-TECH's IS-2, KinetAsyst software or the Kinetic Studio suite. The accessory is available for use with all SF-61 and KinetAsyst dedicated stopped-flow fluorimeters.
Relevant Application Notes:
AN.012.S60
Binding studies of a DNA repair enzyme using stopped-flow fluorescence anisotropy
