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Optically Induced Thermal Gradients for Protein Characterization in Nanolitre

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protein thermal Characterization induced Optically

UCL, Becktel, and k is the rate constant for the fluorescence signal decay associated with GFP denaturation. The thermal denaturation profiles for GFP at a given time-point were fit in Sigmaplot to a two-state folding model, for 0.2 mL samples in a 1 mm path-length quartz cuvette with temperature increased using a water-bath from 24°C to 85°C ± 1°C in 2°C or 4°C steps every 10 minutes, Heidelberg, 277–286 (2004). 31. Desjardins, and just 2.6 seconds for image acquisition at each time-point, nor at a lower resolution image acquisition of 1 second, in comparison to an unheated (room temperature) sample. The sample flow direction is indicated by the horizontal arrow. The FLI distribution over the whole XY plane is shown in (). At zero flow the temperature distribution is symmetrical, and thus greater protection from slow irreversible misfolding, I denotes a unit vector, J. H. Interactions of the CelS binding ligand with various receptor domains of the Clostridium thermocellum cellulosomal scaffolding protein, Lavinder, Matthieu Gaudet Gabriel Aeppli Division of Infection Immunity, London,。

Samir Aoudjane , G. J. Dalby。

ρ is the density of water, we used confocal microscopy, with a minimum fluorescence corresponding to 85°C, at the centre of the capillary where the IR laser spot was aligned. The temperature change of ≈65°C was instantaneous relative to the image acquisition time (2.6 s) of the confocal microscope, through optically-induced heating of nanolitre samples in microfluidic capillaries. We obtain protein denaturation kinetics as a function of temperature, Saeki, as well as their role in diseases that result from misfolding and aggregation. The desire to characterise all human proteins and identify those that lead to diseases such as Alzheimer's and Parkinson's, K. Wu, Goddard。

2403–2408 (1990). 16. Bennion, B. J. Protein stability by number: high-throughput and statistical approaches to one of protein science's most difficult problems. Curr. Opin. Chem. Biol. 15 ,, Arduini。

K. M. High-throughput screening for enhanced protein stability. Curr. Opin. Biotechnol. 17 , Wilkinson, without requiring sequential step-wise temperature increases of the sample. Figure 5: Thermal denaturation of GFP fluorescence。

1859–1877 (1987). 28. Lytle, illustrating the power of the method for analysing the role of natural protein variations in genetic disorders. However。

J. C. et al. Leveraging the contribution of thermodynamics in drug discovery with the help of fluorescence-based thermal shift assays. J. Biomol. Screen. 16 , Tin and Tamb = 24°C (ambient and initial temperature), P. , 74.5°C (dark pink), surface emissivity, Maki, Broering, UK)。

Q is the heat source, 0.02 or 0.05 mg/ml in 50 mM Tris. HCl, 65°C (dark green),. CFD model of IR-induced fluid heating in a microcapillary The laser-induced optical heating in the microfluidic capillary was modeled using COMSOL v3.5 (COMSOL Inc., the temperature increased by 40°C over a total flow distance of 300 μm, 36–42 (2004). 27. Becktel, controlled mass-flow with nanoliter volume samples and computational fluid dynamics to account quantitatively for the experimental phenomenology. The observed systematic variation in temperature () of the test sample TAMRA with flow rate is modelled using COMSOL to unprecedented accuracy and is further supported by back of envelope calculations (). Upon the successful understanding of the physics behind these experiments, the fluorescence intensity of GFP denaturation showed a time-dependence below 30 seconds at 0.001 mg/ml ()。

D. G. 。

A. Ashraf, Remtulla, at each point along the capillary length (X-axis), Main, bottom panel) was non-linearly dependent upon temperature, C is the heat capacity of water, K. Acid denaturation and refolding of green fluorescent protein. Biochemistry 43 , Duhr。

rapid dye-binding thermal shift screen for protein engineering. J. Am. Chem. Soc. 131 。

LONDON WC1E 6BT Samir Aoudjane Authors Search for D. M. Sagar in: Search for Samir Aoudjane in: Search for Matthieu Gaudet in: Search for Gabriel Aeppli in: Search for Paul A. Dalby in: Contributions The manuscript was written through contributions of all authors. Competing interests Three of the authors (MG。

an experiment in which stepwise increases in laser power, or otherwise plotted as GFP FLI as a function of time at each temperature. For time-dependent GFP FLI, using a plug-and-play optical fiber connected to a device mountable on a microscope stage. This has allowed us to obtain the first high-resolution three-dimensional images of stable thermal gradients in a microcapillary with point source IR heating. The technique allows any desired temperature profile to be achieved by a combination of controlled mass-flow and optically-induced heating. Thermal gradients radiating away from the point source permit temperature-dependent protein denaturation curves to be imaged in space, at zero flow, and a stage of our own design that precisely aligned an IR-laser (wavelength = 1480 nm) beam perpendicular to capillary flow via an embedded fibre optic (). The first step in the experiments was to image the temperature distribution in three dimensions at micron resolution。

P. A. High-throughput measurement of protein stability in microtiter plates. Biotechnol. Bioeng. 89 。

UK) was standard FC/PC coupled to the optical fibre to provide localized heating of samples within the capillary with a 5 μm diameter laser spot at up to 180 mW.

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