No technique is perfect, so why not use lots!
Our aim is to probe biological systems to the deepest level of detail that is possible. No single experimental technique in isolation can achieve this and so we use a range of approaches. That means our lab is home to some neat 'toys' and we are grateful to our collaborators for sharing their 'toys' with us. Below are some of the major instruments that we use to give you a flavour of the breadth of approaches that we use.
Stopped-flow is a technique that allows the rapid mixing of reagents and subsequent detection on fast time scales (~ 1000 1/s). We use this technique to follow enyme catalysed ractions and protein conformational change. The data we get out are the kinetics of these processes. Applying simple physical models to the kinteic data allow us to learn fundamental detail about the processes we are studying.
Our stopped-flow is set up to measure absorbance, fluorescence, dual detection emission (FRET) and fluorescence anisotropy in either single mixing or sequential mixing modes.
Increasing the hydrostatic pressure perturbs a pre-exisiting equilibrium of states. We use this technique to study the equilibrium of conformational states that are accessible to proteins and what the thermodynamic 'drivers' are for conformational change.
The fluorescence spectrometer is our work horse instrument allowing rapid scanning before we emmbark on tougher experiments. That said, fluorescence excitation and emission spectra are highly informative, with the magnitude and peak position of fluorophores reflecting fundamental information about their environment. For example, protein tryptophan residues have characteristc emission spectra that inform on how solvent exposed they are and is often used to monitor changes to the tertiary protein structure.
CD is a poweful approach to monitor the secondary structure of proteins. Much of our work is on protein structural disorder and so we tend to monitor in the far-UV region of the spectrum where the realtive proportion of ordered and disordered structure can be assessed. We use the CD instrument in the Pantos group in Chemistry at Bath. This instrument also has the capability for simultaneous CD and fluorescence detection.
Single molecule spectroscopy
Single molecule spectroscopy is a powerful apporach to detect molecular heterogenity and rare states. We use this apporach both to monitor the activity of individual enzymes, but also to capture the range of conformational states a protein can adopt. We either use confocal or TIRF alternating laser excitation spectroscopy, using the instruments at the Research complex at Harwell in the group of Marisa Martin-Fernandez.
3D printing and device fabrication
We seek to exploit fundamental concepts to find new biotechnology solutions. So translate our approaches into the field or a different environement we fabricate devices using 3D printing and microscale optics.