Microscale Thermophoresis - Biozentrum

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  • Microscale Thermophoresis Technology and Applications

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    www.nanotemper.de

    Contents

    1. Technology

    2. Technology Platform

    3. Handling

    4. Applications

    5. Publications

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    www.nanotemper.de

    Microscale Thermophoresis (MST) is a powerful new technology, and easy to handle. It detects changes in the hydration shell of molecules and measures biomolecule interactions under close-to-native conditions: immobilization-free and in bioliquids of choice. Infrared-lasers are used to achieve precise microscale temperature gradients within thin glass capillaries that are filled with a solution of choice (buffer or serum, cell lysate and other bioliquids). Molecules move along these temperature gradients. Extensive research conducted at the Biophysics Department of the Ludwig-Maximilians-University Munich (LMU) identified the solvation entropy and the hydration shell of molecules as the driving force. Any change of the hydration shell of biomolecules due to changes in their primary, secondary, tertiary and/or quaternary structure affects the thermophoretic movement and is used to determine binding affinities with high accuracy and sensitivity. NanoTempers unique technology is ideal for basic research applications requiring flexibility in the experimental scale, as well as for pharmaceutical research applications including small molecules profiling, which are difficult to access with established technologies as they need a high sensitivity.

    The experimental procedure is straightforward and

    eliminates expensive and tedious sample preparation. In

    combination with the capillary format it reduces the

    overall costs and the setup costs which are typically

    associated with standard molecular interaction

    technologies.

    The technology uses fluorescence in combined with IR-Laser optics for local heating of the sample. The heating laser is focused through the same objective used for fluorescence detection. This allows a precise local microscopic heating of the sample within the capillary and simultaneously and observation of local changes of fluorescence intensity due to the motion of labeled molecules in the glass capillaries.

    Microscale Thermophoresis

    1. Technology

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    Fluorescently labeled molecules or particles are initially distributed evenly and diffuse freely in solution. By switching on the IR-Laser, the molecules experience a thermophoretic force in the temperature gradient and typically move out of the heated spot. In the steady state, this molecule flow is counterbalanced by ordinary mass diffusion. After turning off the laser, the particles diffuse back to obtain a homogeneous distribution again. The following stages are recorded for each sample: fluorescence signal before turning the IR laser on, fast temperature-dependent changes in fluorescence intensity, thermophoresis and back diffusion after switching the laser

    off.

    NanoTempers Monolith platform provides instruments, consumables and reagents for analyzing biomolecule interactions with fluorescence and label-free. It utilizes Microscale Thermophoresis to enable real-time, immobilization free analysis of biomolecules providing information on the affinity, stoichiometry and aggregation properties of biomolecules in buffers and complex biological liquids including blood serum and cell lysate. The Monolith NT.115 and NT.LabelFree are NanoTempers instruments for basic research and pharmaceutical applications. The NT.115 uses fluorescence dyes to read out the thermophoretic effect, while the NT.LabelFree uses intrinsic tryptophane fluorescence. Both are based on NanoTempers Microscale Thermophoresis technology. Equipped with the standard sample tray, each instrument can process automatically up to 16 samples per run in 10 minutes.

    Monolith Series Instruments

    2. Technology Platform

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    Key Benefits

    Microscale Thermophoresis can monitor the binding of single ions (40Da) or small molecules (300Da) to a target as well as the binding of ribosomes (2.5MDa). Microscale Thermophoresis is easy to handle and allows to measure the binding of biomolecules as well as the activity of enzymes. It is ideal for basic research applications requiring flexibility in the experimental scale, as well as for pharmaceutical research applications, including small molecules profiling, which are difficult to access with established technologies as they require a high sensitivity.

    measure affinities (KD, dissociation constant) between any (bio)molecules directly in bioliquids

    study membrane bound proteins directly in liposomes or in detergent solutions

    study multi component reactions, complex formation, order of assembly and interfering factors

    study the effect of serum, cell lysate or other bioliquids on biomolecules

    separate aggregation and other artifacts from true binding events

    measure with fluorescence label and label-free

    access larger screening projects in a label-free manner using fluorescently labeled tool compounds

    discriminate between different binding sites on a target of interest

    study the stoichiometry and determine the number of binding sites of biomolecules

    study the binding energetics dG (free energy ), dH (enthalpy) and dS (entropy)

    study the inhibitor affinity, Ki either directly or in a competition experiment

    Microscale Thermophoresis monitors binding and biochemical activity of biomolecules under close-to-native conditions:

    immobilization-free

    label-free

    In a solution of choice, ranging from standard and proprietary buffers to complex bioliquids including blood serum or cell lysates

    at a temperature of choice

    Unmatched Sensitivity

    Unique performance

    Close-to-native conditions

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    Monolith NT.115

    LED 1 /nm

    LED 2 /nm

    Blue Dyes Green Dyes Red Dyes

    NT.115 Blue/Green

    Ex:470

    Em:520

    Ex:550

    Em:600

    FITC/FAM/GFP/YFP Cy3/RFP/mCherry no detection

    NT.115 Blue/Red

    Ex:470

    Em:520

    Ex:625

    Em:680

    FITC/FAM/GFP/YFP no detection Cy5/Alexa647

    NT.115 Green/Red

    Ex:520

    Em:570

    Ex:625

    Em:680

    YFP

    Cy3/RFP Cy5/Alexa647

    The capillary format is inexpensive, easy to handle and offers maximum flexibility in the experiment scale. The sample tray format allows to process automatically up to 16 capillaries (e.g. for a detailed KD-analysis), or alternatively to perform smaller pilot experiments or end point measurements involving 2-3 capillaries only. Microscale Thermophoresis requires approximately 100fold less sample material compared to standard technologies. Capillary volume: 3-5 l at concentrations as low as 1 nM of the labeled molecule. The Monolith NT.115 instrument is supported by a software for data acquisition and analysis. 3 models of the Monolith NT.115 instrument are offered, which differ in the excitation/detection spectrum to detect blue, green and/or red fluorescent dyes, as defined by the respective color of the excitation light.

    Capillary format

    Requires little sample material

    Dedicated Data Acquisition and Analysis Software

    Monolith Models

    Monolith NT.115

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    Monolith NT.LabelFree

    LED 1 /nm

    Molecules (examples)

    NT.Label Free

    Ex:280

    Em:360

    Proteins containing Tryptophane 2-Aminopurin

    8-vinyl-deoxyadenosine BIRB-796

    The Monolith NT.LabelFree instrument has an excitation wavelength of about 280nm and an emission wavelength around 360nm. It allows to use any molecule that has a fluorescence in this range to be used as the labeled constituent of a MST experiment (e.g. tryptophane, 2-Aminopurin, 8-vinyl-deoxyadenosine, etc)

    NanoTemper offers kits and capillaries for use with

    Microscale Thermophoresis that enable you to get high

    quality MST results. The products are specially designed

    for the MST instruments NT.115 and NT.LabelFree. NanoTemper provides you with capillaries that fullfill the high requirments of MST in terms of reproducibility, glass and surface quality as well as background fluorescence. The capillaries come with different surface coatings to stabilize even the most complex samples in solution.

    Monolith NT.LabelFree

    Capillaries

    Monolith Consumables

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    Our labeling kits contain fluorescent dyes that are widely tested with MST. The labeling protocol ensures good labeling efficiency and purification. NanoTemper offers dyes that are optimized for protein compatibility and MST Temperature Jump. The fluorescence emission and detection fits perfect to the BLUE, GREEN and RED channel in the NT.115 instruments.

    Our Assay Development and Control Kits allow you to

    get started with MST quickly and train new lab members.

    Assay Development and Control Kits

    Labeling Kits

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    Microscale Thermophoresis is easy to handle and

    involves the following steps:

    One of the binding partner is labeled using standard

    fluorescent labeling protocols. Blue, green or red dyes

    and all coupling chemistries are compatible with our

    technology. NanoTemper provides dyes optimized for

    protein compatibility and MST analysis. In case the

    Monolith NT.LabelFree is used, no labeling is necessary.

    A titrati