How does ThermoFluor work?

Background: The ThermoFluor assay is a quick, temperature-based assay to assess the stability of proteins. The method takes a small amount of protein (45 uL of 5 uM) and adds SYPRO Orange dye. The dye is similar to ANS and binds to hydrophobic patches/denatured protein/molten globules and fluoresces.

What does a thermal shift assay tell you?

A thermal shift assay (TSA) measures changes in the thermal denaturation temperature and hence stability of a protein under varying conditions such as variations in drug concentration, buffer pH or ionic strength, redox potential, or sequence mutation.

What is DSF assay?

Differential scanning fluorimetry (DSF), also known as ThermoFluor or Thermal Shift Assay, has become a commonly-used approach for detecting protein-ligand interactions, particularly in the context of fragment screening.

What is CPM assay?

A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.

How is protein stability measured?

Methods of Determining Protein Stability

  1. Determining Protein Stability: Some of the Most Common Methods Used.
  2. Differential Scanning Calorimetry (DSC)
  3. Pulse-Chase Method.
  4. Bleach-chase method.
  5. Cycloheximide-chase method.
  6. Circular Dichroism (CD) Spectroscopy.
  7. Fluorescence-based Activity Assays.

What is the most common dye used for differential scanning fluorimetry analysis?

The Sypro orange dye commonly used for DSF revealed multiple overlapping thermal protein denaturation transitions for both the mAb and the Fab fragment, making quantitative analysis of ligand binding by thermal stabilization problematic.

What is thermal fluorescence?

Also known as ThermoFluor, differential scanning fluorimetry (DSF) or temperature-dependent fluorescence (TdF), FTSA exploits the phenomenon whereby the binding of a ligand enhances the thermal stability of its target protein, this phenomenon can be used to provide real-time information with respect to compound-target …

How do you calculate Tm of fluorescence?

In the HMD method, the Tm is determined by calculating the temperature of HMD (DT50), which is the temperature at which the fluorescence intensity reaches 50% of the highest fluorescence intensity. If no protein or a denatured protein is added, the returned Tm is the initial temperature (32°C in the exemplar data).

Are proteins stable?

The net stability of proteins is quite small and is the difference between two large opposing forces. Proteins from thermophilic organisms exhibit high thermal stability, and they proliferate at temperatures around 80◦C to 100◦C.

What is differential scanning fluorimetry?

Differential Scanning Fluorimetry (DSF) measures protein unfolding by monitory changes in fluorescence as a function of temperature. Conventional DSF uses a hydrophobic fluorescent dye that binds to proteins as they unfold. NanoDSF measures changes in intrinsic protein fluorescence as proteins unfold.

Which are the variant of fluorimetry instrument?

There are two basic types of fluorometers: the filter fluorometers and spectrofluorometer. The difference between them is the way they select the wavelengths of incident light; filter fluorometers use filters while spectrofluorometers use grating monochromators.

What would happen in a protein thermal stability assay If your protein has a lot of hydrophobic residues on its surface?

When the protein unfolds, the exposed hydrophobic surfaces bind the dye, resulting in an increase in fluorescence by excluding water. Detergent micelles will also bind the dye and increase background noise dramatically.

How does differential scanning fluorimetry work?

What is melting point in differential scanning fluorimetry?

Differential scanning fluorimetry (DSF) uses a dielectric sensitive fluorescent dye to measure the thermal denaturation, or “melting” point (Tm) of a protein under different conditions, in this case in the absence and presence of a candidate ligand.

Which interaction is the most important for protein stability?

The experimental results from many groups over the past few decades have confirmed that hydrophobic interactions do make the major contribution to protein stability but that hydrogen bonds also make a large contribution.

What affects protein stability?

Many factors affect the process of protein folding, including conformational and compositional stability, cellular environment including temperature and pH, primary and secondary structure, solvation, hydrogen bonding, salt bridges, hydrophobic effects, van der Waals (vdW) forces, ligand binding, cofactor binding, ion …

What is protein stability analysis?

The protein stability analysis assists to determine the stability of proteins in a sample. There are various methods and techniques available to determine protein stability. This analysis is carried out by biotechnological and pharmaceutical industries for drug development and by research and academic institutes.

How do you measure the melting temperature of a protein?

Currently protein melting temperature is determined by laboratory methods such as Differential Scanning Calorimetry, Circular Dichroism, Fourier transform infrared spectroscopy and several other methods. These methods are laborious and costly.

How does temperature affect protein stability in solution?

The larger and more positive G u , the more stable is the protein Therefore, for most proteins, as the temperature increases, the ∆G u decreases and becomes zero at equilibrium Tm, where [ folded ] = [ unfolded ] Therefore, protein will be most stable at the conditions where Tm is the highest Protein stability in solution is influenced by:

Which buffer is more stable for thermal shift assay?

Here, the protein is significantly more stable in 50 mM sodium phosphate buffer, pH 7.0 than several others at the same concentration and pH, including: L-Arg/L-Glu, Tris, HEPES, and MOPS. This unit describes the full workflow for a thermal shift assay, starting with a buffer screen to optimize protein stability.

How to monitor the thermal denaturation of SYPRO orange protein?

Through a systematic increase in temperature and concomitant monitoring of SYPRO Orange fluorescence emission, it is possible to monitor thermal denaturation of the protein in many conditions simultaneously.

What is the workflow for a thermal shift assay?

This unit describes the full workflow for a thermal shift assay, starting with a buffer screen to optimize protein stability. Special focus is given to the crucial step of data analysis to derive melting temperatures from the raw thermal denaturation data (Support Protocol 1).