#  MicroScale Thermophoresis (MST) 

 



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[**CMI MST resources and user guide**](/node/750986#mst-resources)**⬇︎**

[MicroScale Thermophoresis](http://www.nanotemper-technologies.com/technologies/mst-technology/) (MST) is an immobilization-free technology for measuring biomolecular interactions. The MST instrument detects the motion of fluorescent molecules along a microscopic temperature gradient, which reflects changes in the molecular hydration shell, charge, or size. Since one or all of these parameters will change with virtually every binding event, a wide range of biomolecules can be measured, from ions and small molecule fragments to large macromolecular complexes, in small volumes (~20 μl), in a wide range of standard buffers and complex mixtures such as liposomes, detergent, serum, and cell lysates.

 ![MST experimental setup diagram](/sites/g/files/omnuum4016/files/2025-01/MST_diagram_design.jpg)

 

   ![Monolith NT.115  Image](/sites/g/files/omnuum4016/files/styles/hwp_1_1__360x360_scale/public/2025-01/NT115_01.png?itok=6AtJv0Sr) 

 

The CMI has a [Monolith NT.115pico](https://nanotempertech.com/monolith/) from NanoTemper Technologies.

**Key Features**

- fast measurement: Kd in about 10 min
- wide Kd range from pM/nM to mM range
- immobilization free, in-solution measurements
- low sample consumption: volume (10 µl per concentration)
- wide size range for interactants (from ions to MDa complexes)
- measurements in complex mixtures (cell lysates, serum, detergents, liposomes)



 

 MST Resources Sample Preparation Required Supplies NT.115 Detectors 

## MST Resources

[CMI Monolith MST Getting Started Guide](/sites/g/files/omnuum4016/files/2025-03/CMI%20Monolith%20MST%20Getting%20Started%20Guide_0.pdf "CMI Monolith MST Getting Started Guide")

[MicroScale Thermophoresis technology](http://www.nanotemper-technologies.com/technologies/mst-technology/), from NanoTemper Technologies

 

 

 

## Sample Preparation

**Assay Buffers**

- Many buffers are compatible with MST. It’s usually a good idea to start with a buffer system in which your proteins are well behaved.
- **Addition of 0.05% Tween 20 or other detergent is usually required** to prevent sticking of proteins to the capillaries.
- Each capillary should be prepared with identically matched buffer.
- **Assay buffer** (with detergent) is used to dilute the fluorescent molecule to 2X.
- **Ligand buffer** is used to dilute the ligand and should match the highest concentration of ligand
- 0.5-1 mg/ml BSA can also be used to minimize non-specific binding.
- Buffer cannot be opaque.
- High viscosity samples may be hard to fill (up to 10% glycerol is fine).

**Samples**

- All MST experiments are setup with one fluorescently-labeled molecule (the **Target**) at a fixed concentration mixed with various concentrations of a non-fluorescent molecule (the **Ligand**).
- Concentration should be accurately measured 
    - Errors in Target concentration can affect fluorescent signal and may affect the fit
    - Errors in the Ligand concentration will directly translate to errors in the KD
- Protein aggregates will interfere with MST
    - Filter or centrifuge samples before use.
    - Assess protein heterogeneity via light scattering.
    - Purify protein samples with soluble aggregates by size-exclusion chromatography.

**Target Sample** (the fluorescent molecule)

- ~200 µL/titration at >2X working concentration
- 5-20 µM unlabeled protein, if using a chemical labeling kit
- RED detector:
    - Stock concentration of labeled Target: ≥ 10 nM
    - Recommended working concentration: 5 nM (for KD > nM)
    - Minimal working concentration: ≥ 50 pM (used for KD in pM range)
- BLUE detector:
    - Stock concentration of labeled Target: ≥ 40 nM
    - Recommended working concentration: 20 nM
    - Minimal working concentration: ≥ 5 nM

***Ligand Sample*** (the non-fluorescent binding partner)

- ~ 20 µL/titration, at 2X working concentration (bring the highest stock concentration available for an unknown KD)
- Recommended stock concentration ≥ 100X the expected KD
- Recommended working concentration ≥ 50X KD

 

 

 

## Required Supplies

- fluorescent target sample and non-fluorescent ligand sample and matched buffer
- MST capillaries (see below)
- 0.2 ml tubes for sample preparation (provided by CMI)
- pipetors and tips for liquid handing

[**NanoTemper Supplies**](https://shop.nanotempertech.com/)

**MST Capillaries**

- Monolith NT.115 Standard Treated Capillaries, MO-K022 (available from the CMI, at cost)
- Monolith NT.115 MST Premium Coated Capillaries, MO-K025 (available from the CMI, at cost)

 **Labeling Kits (optional)**

- NanoTemper Protein Labeling Kit RED-NHS 2nd Generation (Amine Reactive), MO-L011
- NanoTemper Protein Labeling Kit RED-MALEIMIDE 2nd Generation (Cys Reactive). MO-L014
- NanoTemper His-Tag Labeling Kit RED-tris-NTA 2nd Generation, MO-L018

 

 

 

## NT.115 Detectors

**Pico RED detector**

- excitation wavelength: 600-650 nm
    - eg. AlexaFluor647, NT647, Cy5
- fluorophore concentration ≥ 50 pM
- Kd range: pM - mM

**Nano BLUE detector**

- excitation wavelength 460-490 nm
    - eg. fluorescein, AlexaFluor488, NT495, GFP
- fluorphore concentration ≥ 5 nM
- Kd range: nM - mM

   ![Monolith Blue Red filtners](/sites/g/files/omnuum4016/files/styles/hwp_1_1__360x360_scale/public/2025-02/Monolith%20NT.115_BLUE_RED_filters.png?itok=o4MEeQ9a) 

 

 

 

 

 

 

 [ Data Management and File Formats arrow\_circle\_right ](/data-management) 

 

 

 

 

 



 

 See also:- [ molecular interactions ](/cmi-terms/molecular-interactions)
- [ MicroScale Thermophoresis ](/cmi-terms/microscale-thermophoresis)