Microscale Thermophoresis Technology and Applications
2. Technology Platform
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
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.
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
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
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:
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
LED 1 /nm
LED 2 /nm
Blue Dyes Green Dyes Red Dyes
FITC/FAM/GFP/YFP Cy3/RFP/mCherry no detection
FITC/FAM/GFP/YFP no detection 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.
Requires little sample material
Dedicated Data Acquisition and Analysis Software
LED 1 /nm
Proteins containing Tryptophane 2-Aminopurin
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.
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
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.