one for water (the control) and one for the sample. The sensor detects temperature
difference between the cells. The ligand, loaded by a syringe in a cell sample, binds
the molecule, and the heat change is measured compared to the control.
This technique has several advantages: (1) the protein can be left in solution and
no molecule modification is therefore required, and it measures the affinity of
proteins in their native state; (2) the addition of chemofluorescent tags is not
necessary; (3) stoichiometry, association constant and binding enthalpy can be
characterized in a single experiment; and (4) experiments can be performed in
systems such as dispersions, intact organelles or cells. The main disadvantage of
ITC is the limited range for measuring binding affinities (association constants from
10
4 to 10
À1 M). In addition, it is a slow technique for HTS analysis (0.25–2 h/assay).
In recent years, new technologies for a more rapid analysis of data have been
designed. In 2017, Mitter et al. described the development of a new ITC technique
allowing for the acquisition of data in the order of 0.2 s and less.
ITC is widely used for:
• Quantification of binding affinity
• Confirmation of binding targets of small molecules and characterization of
mechanisms of action
• Validation of IC50 and EC50
• Measurement of enzyme kinetics
ITC can be performed using MicroCal™ (Malvern Panalytical) ITC instruments
such as MicroCal PEAQ-ITC, which gives high sensitivity and quality data with low
sample consumption, or MicroCal VP-ITC, an easy-to-use isothermal titration
calorimeter. ITC is mainly used for target validation, measuring enzyme kinetics,
investigating the binding mechanisms and potency of drugs and studying the design
of inhibitors.
1.1.4 Microscale Thermophoresis (MST)
MST, similarly to ITC, quantifies protein binding in a temperature gradient, but the
movement of the molecule and conformational state is recorded by the use of
fluorophores [16, 17] (Fig. 2). Infrared laser is used for temperature increase. The
molecule is placed in a capillary containing a fluorescent solution, and an infrared
ray of a wavelength of 1,480 nm is projected against the capillary, causing an
increase in temperature. The fluorophores with an increased temperature are excited
and emit fluorescence. The ability to emit fluorescence by temperature increase is
inversely proportional to the ability to bind the ligand. MST analysis has no
limitations in terms of molecule size or molecular weight and is performed in a
matter of minutes in free solution.
There are a number of different devices to monitor MST, such as the Monolith
NT.Automated or Monolith NT115Pico (NanoTemper) instruments designed to
detect even low picomolar concentrations in microlitre volumes of red-emitting
fluorophores. With minimal sample consumption, they provide high sensitivity
Advanced Assays in Epigenetics
529
difference between the cells. The ligand, loaded by a syringe in a cell sample, binds
the molecule, and the heat change is measured compared to the control.
This technique has several advantages: (1) the protein can be left in solution and
no molecule modification is therefore required, and it measures the affinity of
proteins in their native state; (2) the addition of chemofluorescent tags is not
necessary; (3) stoichiometry, association constant and binding enthalpy can be
characterized in a single experiment; and (4) experiments can be performed in
systems such as dispersions, intact organelles or cells. The main disadvantage of
ITC is the limited range for measuring binding affinities (association constants from
10
4 to 10
À1 M). In addition, it is a slow technique for HTS analysis (0.25–2 h/assay).
In recent years, new technologies for a more rapid analysis of data have been
designed. In 2017, Mitter et al. described the development of a new ITC technique
allowing for the acquisition of data in the order of 0.2 s and less.
ITC is widely used for:
• Quantification of binding affinity
• Confirmation of binding targets of small molecules and characterization of
mechanisms of action
• Validation of IC50 and EC50
• Measurement of enzyme kinetics
ITC can be performed using MicroCal™ (Malvern Panalytical) ITC instruments
such as MicroCal PEAQ-ITC, which gives high sensitivity and quality data with low
sample consumption, or MicroCal VP-ITC, an easy-to-use isothermal titration
calorimeter. ITC is mainly used for target validation, measuring enzyme kinetics,
investigating the binding mechanisms and potency of drugs and studying the design
of inhibitors.
1.1.4 Microscale Thermophoresis (MST)
MST, similarly to ITC, quantifies protein binding in a temperature gradient, but the
movement of the molecule and conformational state is recorded by the use of
fluorophores [16, 17] (Fig. 2). Infrared laser is used for temperature increase. The
molecule is placed in a capillary containing a fluorescent solution, and an infrared
ray of a wavelength of 1,480 nm is projected against the capillary, causing an
increase in temperature. The fluorophores with an increased temperature are excited
and emit fluorescence. The ability to emit fluorescence by temperature increase is
inversely proportional to the ability to bind the ligand. MST analysis has no
limitations in terms of molecule size or molecular weight and is performed in a
matter of minutes in free solution.
There are a number of different devices to monitor MST, such as the Monolith
NT.Automated or Monolith NT115Pico (NanoTemper) instruments designed to
detect even low picomolar concentrations in microlitre volumes of red-emitting
fluorophores. With minimal sample consumption, they provide high sensitivity
Advanced Assays in Epigenetics
529