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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.
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