Protein Thermal Shift Software & Reagents

Protein Thermal Shift™ solution for differential scanning fluorometry

Applied Biosystems® Protein Thermal Shift™ Software and reagent kits, plus real-time PCR systems, offer high-throughput protein melt analysis that requires <1 µg of sample per well, at a cost that is significantly lower than alternative methods.

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Protein Thermal Shift™ Overview

Proteins are typically key target molecules studied during the drug development process. The high-throughput screening of small molecule and ligand libraries that bind to protein targets is often an important and time-consuming part of the process—requiring the screening of thousands of samples with a variety of assays over a period of months. However, since protein targets can be challenging to work with due to their susceptibility to degradation and aggregation, protein stability screening is often an important component of lead-generation programs.

Protein stability screening can be performed using the protein melting method often employed in research programs that study native proteins. Protein melting is useful for identifying ligand and buffer conditions that maximize the stability of proteins during purification, crystallization, and functional characterization.

Historically, protein melt screening methods have been inefficient or expensive, either capable of analyzing only one sample at a time, or confined to high-throughput methods that required milligram quantities of protein sample and incurred high reagent costs. Our Protein Thermal Shift™ Software and reagent kits offer an economical and efficient protein method for melt analysis, with the capability of high-throughput analysis using very small sample quantities to identify ligands, mutations, modifications, and buffer conditions that increase their Tm and relative stability. They can also be used to screen antibody–ligand binding as part of the antibody optimization process.

The Protein Thermal Shift™ Assay Method

Protein stability changes with buffer pH, salt content, and the presence of various co-factors in storage or reaction buffers. Real-time melt experiments that use protein-binding dyes, such as PTS, and our real-time PCR systems, yield a fluorescence profile that is specific to the protein of interest in a given test buffer environment. Variations in pH, salt content, or test buffer components appear as changes in this fluorescence profile (melt curve). This is converted to a Tm which is calculated based on the inflection point of the melt curve.

  1. Mix protein, buffer, ligand (if applicable), and PTS dye
  2. Run a melt-curve experiment on a real-time PCR instrument
  3. The protein unfolds as it is heated
  4. The environmentally-sensitive PTS dye binds exposed hydrophobic regions and fluoresces
  5. The melting temperature (Tm) is calculated from the melt curve
  6. Changes in Tm are correlated to changes in protein stability or ligand binding
Protein Thermal Shift™ Applications

Protein Stability Screening:

  • Improving protein preparation (buffer, pH, salt, excipients)
  • Profiling conditions that favor crystallization
  • Developing protein formulations and storage buffers
  • Analyzing effects of mutations or modifications
  • Performing protein preparation quality control

High-Throughput Ligand Screening:

  • Small-molecule and fragment library screens
  • Antibody-target specificity
  • Protein-protein interactions
  • Inhibitor binding screening


Enlarge Image

A Protein Thermal Shift ™ Profile.

Overview
Protein Thermal Shift™ Overview

Proteins are typically key target molecules studied during the drug development process. The high-throughput screening of small molecule and ligand libraries that bind to protein targets is often an important and time-consuming part of the process—requiring the screening of thousands of samples with a variety of assays over a period of months. However, since protein targets can be challenging to work with due to their susceptibility to degradation and aggregation, protein stability screening is often an important component of lead-generation programs.

Protein stability screening can be performed using the protein melting method often employed in research programs that study native proteins. Protein melting is useful for identifying ligand and buffer conditions that maximize the stability of proteins during purification, crystallization, and functional characterization.

Historically, protein melt screening methods have been inefficient or expensive, either capable of analyzing only one sample at a time, or confined to high-throughput methods that required milligram quantities of protein sample and incurred high reagent costs. Our Protein Thermal Shift™ Software and reagent kits offer an economical and efficient protein method for melt analysis, with the capability of high-throughput analysis using very small sample quantities to identify ligands, mutations, modifications, and buffer conditions that increase their Tm and relative stability. They can also be used to screen antibody–ligand binding as part of the antibody optimization process.

Method
The Protein Thermal Shift™ Assay Method

Protein stability changes with buffer pH, salt content, and the presence of various co-factors in storage or reaction buffers. Real-time melt experiments that use protein-binding dyes, such as PTS, and our real-time PCR systems, yield a fluorescence profile that is specific to the protein of interest in a given test buffer environment. Variations in pH, salt content, or test buffer components appear as changes in this fluorescence profile (melt curve). This is converted to a Tm which is calculated based on the inflection point of the melt curve.

  1. Mix protein, buffer, ligand (if applicable), and PTS dye
  2. Run a melt-curve experiment on a real-time PCR instrument
  3. The protein unfolds as it is heated
  4. The environmentally-sensitive PTS dye binds exposed hydrophobic regions and fluoresces
  5. The melting temperature (Tm) is calculated from the melt curve
  6. Changes in Tm are correlated to changes in protein stability or ligand binding
Applications
Protein Thermal Shift™ Applications

Protein Stability Screening:

  • Improving protein preparation (buffer, pH, salt, excipients)
  • Profiling conditions that favor crystallization
  • Developing protein formulations and storage buffers
  • Analyzing effects of mutations or modifications
  • Performing protein preparation quality control

High-Throughput Ligand Screening:

  • Small-molecule and fragment library screens
  • Antibody-target specificity
  • Protein-protein interactions
  • Inhibitor binding screening


Enlarge Image

A Protein Thermal Shift ™ Profile.

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Literature
Protein Thermal Shift™ software
Protein Thermal Shift software

Protein Thermal Shift™ Software was developed for analysis of protein melt fluorescent readings directly from Applied Biosystems® real-time PCR instrument files. Different proteins will have different PTS™ profiles, each with a unique melt curve shape, slope, signal-to-noise ratio, and temperature melt range. The Protein Thermal Shift™ Software generates one or multiple Tm values from these curves by the following two methods: Boltzmann-derived Tm and the Derivative curve determined Tm. The software makes it easy to quickly compare the shift in Tm (delta Tm or ΔTm) between different assay conditions or ligands to a reference sample, providing a tool to screen and identify conditions that stabilize (or destabilize) a protein or ligands that bind to the protein of interest.

Protein Thermal Shift™ assay

Protein Thermal Shift™ (PTS) reagents enable protein melt assays that can be used as an efficient screening tool for measuring protein thermal stability, identifying suitable buffer conditions, and measuring protein–ligand interactions. The assay is very easy to set up and run, with typical time-to-results of 0.5–2 hours, depending on the real-time system and assay conditions.