Much of the instrumentation in the lab is custom-built to address a specific question. Below are some of the instruments we have put together. We also typically write our own data acquisition and analysis software.
- In order to probe the conformation, dynamics, oligomeric state and kinetics of biological macromolecules we have built a custom single-molecule confocal microscope with time-correlated single-photon capabilities. The ‘scope has a number of features that make it convenient for probing protein-protein interactions:
- 488 nm picosecond diode laser excitation
- 594 nm CW laser excitation
- idQuantique ultra-low-noise SPAD detectors with up to 4 channel capability (currently 2 are implemented)
- Multi-SPC-150 TCSPC electronics
- Fully automated
Time-resolved fluorescence and lifetime-resolved FRET
Time-resolved fluorescence using time-correlated single-photon counting is a versatile tool for biophysical studies. By resolving the nanosecond timescale decay of fluorescence we can obtain information about the local environment of fluorophores (via time-resolved anisotropy, for example) and, with suitable donor and acceptor fluorophores, distance distributions in biological macromolecules. By resolving the lifetime one can also discern sub-populations in heterogeneous samples. For example, a marginally stable protein or peptide may populate both a high-FRET and a low-FRET state. This will be very apparent in the
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Microfluidics and Spectroscopy for Microsecond Folding Kinetics
Protein folding events occur on a wide range of timescales. These are highlighted below with the various techniques used to probe them. One of the limiting factors in protein folding studies has been access to the sub-millisecond timescale for kinetic measurements. Following the lead of the Rousseau, Eaton and Roder groups, we have
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CryoEM grid preparation
Recent advances in cryo-electron microscopy (cryoEM), such as the development of direct detectors, automation and 3D particle reconstruction algorithms, have transformed structural biology by enabling investigators to obtain near atomic resolution structures without the need to grow crystals. A significant challenge that limits the wider applicability of cryoEM as a structural tool is the preparation of suitable samples in thin (< 100 nm) layers of vitreous ice in the approximately micron-sized holes of a grid substrate. This arises from the large surface-area-to-volume ratio of the thin aqueous layers prior to freezing of the thin liquid layer in a cryogen. Biological macromolecules preferentially localize to the air-water interface in the thin liquid layer, giving rise to preferred orientations or, in some cases, denaturing.
Overview: Using 3 Zaber stepper motors and a Hamilton Microlab 500 series syringe pump we’ve assembled a home-built autosampler and written custom software for it. The software is written in Labview (originally version 8.2 but currently in Labview 2016) and the software is available here (you’ll need the LabVIEW Runtime Engine to run the executable). We’ve used the unit for automated titrations (urea and guanidineHCl denaturation of proteins) using time-resolved fluorescence and small-angle x-ray scattering (SAXS) and obtained data that would have been very difficult and laborious to obtain manually. Many thanks to Sagar Kathuria for helping