Microscale Thermophoresis exploits the directed motions of biomolecules and macromolecular complexes in solution in microscopic thermal gradients.Ìý It can be used to measure equilibrium binding events.
Thermophoretic mobility is dependent on the solvation entropy, charge, size and conformation of the molecules observed. Essentially, changes in thermophoretic mobility result from changes in the hydration shell; such changes manifest from almost any binding event, as this will change structure/conformation, size, charge, and so on.Ìý During most binding events one or several of these properties will change, making MST a powerful technique for the quantitative analysis of binding reactions.Ìý Thermophoretic movement is detected via a fluorescently labeled binding partner, and an infrared laser (1480 nm wavelength) is focused onto the sample spot where fluorescence intensity is measured. The focused infrared radiation creates a highly localized temperature gradient (ca. 2 – 8 degrees) in the aqueous sample solution.Ìý In this way, thermophoresis is induced in the sample.Ìý Typically, this results in a rapid drop in initial fluorescence followed by diffusion-limited thermophoresis that lasts several seconds.Ìý These processes are affected by binding events.Ìý When the IR laser is turned off, back-diffusion occurs and fluorescence recovers toward its initial level.
Using MST, you are not only able to determine affinities, but you can also assess other physical parameters such as stoichiometry, aggregation, enthalpy (van’t Hoff plot), slow enzyme kinetics Ìýand oligomerization.
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Exciting Possibilities in Biology, Biochemistry and Biophysics:
Kd in the nM to mM range under near physiological condition ((protein-protein, protein-ligand, DNA/RNA-ligand, DNA/RNA-protein, protein-lipids, peptide-lipids, small molecule binding, membrane proteins in liposomes/nano discs or detergent solution ...)
Determine stoichiometry and number of binding sites
Investigate oligomerization and aggregates
Binding energetics ΔG (free energy), ΔH (enthalpy) and ΔS (entropy)
No immobilization necessary
Either component can be labelled and the label can be located far from the interaction site
Users have to receive hands-on training, from Dr.Erbse, before they are allowed to use the MST independently. Interested users should contact Dr. Erbse to schedule training.
Reading the protocolsÌýdoes not replace the training provided by Dr.Erbse.
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After your training is completed, you will be invited to join the MST Google Calendar.Use the MST Google Calender to sign up for instrument time, include your name and lab.
The MSTÌýis located in the basement of JSCBB in the C-Wing, room C1B60, on East Campus. Proxcard access is required at all times.
Regular user groups are expected to opt into the pool and pay an monthly share that goes towards the general up keep of the Shared Instruments Pool. For detailed information please contact Dr. Annette Erbse.
Users are expected to provide all consumables specific to their experiment themselves, including capillaries.
Users are expected to pay for repairs or parts necessary because of damage caused by carelessness or neglect.
Cost for necessary repairs/services because of normal wear will be split depending on time used between user groups.
Please note - Since repairs/service from normal wear are rare, assuming the instrument is treated correctly, the costs for repairs or service might come a long time after use.
Our MST instrument is a Nano-BLUE/RED Monolith NT.115 from NanoTemper Technologies, equipped with two LED-filter combinations, blue (excitation 450–480 nm, emission 515–570 nm) and red (excitation 600–645 nm, emission 670–730 nm).
