The ISMB Biophysics Centre
at Birkbeck

Initial Characterisation

First questions to answer about a newly purified macromolecule are often whether it is monomeric and whether it is folded. AUC is the method of choice to determine the oligomeric state, and circular dichroism can give a quick, or a detailed answer to questions of fold. For initial tests of enzyme activity and turnover reactions there is a UV/Vis spectrometer available.

Label-free Interaction Analysis

The affinity of a complex is another question AUC is able to address. Surface plasmon resonance (Biacore) monitors association and dissociation reactions with a setup that is ideal for screening. However, isothermal titration calorimetry (ITC) gives the most comprehensive analysis of the thermodynamics of a binding reaction.

Structure and Stability

The differential scanning calorimeter (DSC) measures the stability of a structure by melting it. Sophisticated thermodynamic analyses are possible to determine more than just the melting temperature. CD spectroscopy reveals secondary structure content of proteins and is used to confirm foldedness or to measure the melting temperature as alpha and beta secondary structures transform into random coil.

Between Structure and Function

Fluorescence is intrinsic to many proteins through their aromatic amino acids. Lacking those, labels can be placed specifically to give local information on associations or conformational changes or monitor changes in size of macromolecular complexes by anisotropy. Our fluorescence (and phosphor) imager can analyse and quantitate signals in 2D samples like gels, blots and TLC plates, e.g. to detect complex formation in native gels.

FTIR spectroscopy detects characteristic signatures in the infrared range originating from molecular vibrations. It has a huge range of applications from structural biology and analytical biochemistry to medical diagnostics.

Resolution in Time

Kinetic analyses yield far more information than just KM and kcat. Measuring time courses with millisecond precision can be vital for understanding function, regulation, interactions or mechanics of a macromolecular complex. Our stopped flow measures absorption or fluorescence. The quench flow yields time courses of product formation.





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