Protein Thermal Shift Software & Reagents

Applied Biosystems® Protein Thermal Shift™ (PTS) 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™ 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
 


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A Protein Thermal Shift ™ Profile.

 Proteins are typically the key molecule studied as the drug target for drug development generation. High throughput screening of small-molecule and ligand libraries that bind to protein targets is an important part of the process—requiring screening of thousands of small molecules and ligands with a variety of different assays, requiring months of time. Protein targets are challenging to work with due to their susceptibility to degradation and aggregation, so protein stability screening is often an important component of lead generation programs. Protein stability screening, performed using the protein melting method is employed in other research programs that involve native proteins. Protein melting is an extremely useful screening method for the identification of ligands and/or solution (buffer) conditions that maximally stabilize a protein as part of protein purification, crystallization, and functional characterization.

Historically, the methodologies to perform protein melt screening are either very slow and tedious, analyzing one sample at a time—or if high-throughput, require milligram amounts of protein sample and incur high costs in either reagents, or protein samples, or both. Our Protein Thermal Shift™ (PTS) Software and Reagent kits offer economical protein melt analysis with both high throughput and very small sample quantities. PTS™ Software and reagents enable high throughput screening of proteins to identify ligands, mutations/modifications, or buffer conditions that increase their melting temperature (Tm) and relative stability. They can also be used to screen antibody-ligand binding as part of antibody development.

 

Protein Thermal Shift™ Software

   

Protein Thermal Shift™ Software was developed for analysis of protein melt fluorescentreadings 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.
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Protein Thermal Shift™ Assay

   The 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, and time to result typically takes from 0.5–2 hours, depending on the real-time system and run conditions utilized for the assay.
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How Does a Protein Thermal Shift™ Assay Work?

  • Mix protein, buffer, ligand (if applicable) and PTS™ dye
  • Run a melt curve experiment on a real-time PCR instrument
  • The protein unfolds as it is heated
  • The environmentally-sensitive PTS dye binds exposed hydrophobic regions and fluoresces
  • The Melting Temperature (Tm) is calculated from the melt curve
  • Changes in Tm are correlated to changes in protein stability or ligand binding

 

Protein stability changes with buffer pH, salt content and the presence of various co-factors in a protein’s storage or reaction buffer environment. A real-time melt experiment with a protein binding dye such as the PTS™ Dye, run on any Applied Biosystems® real-time PCR systems, yield a fluorescence profile that is specific to the protein of interest in a given test buffer environment. Variations in the pH, salt content or components of the test buffer, are reflected in changes in this fluorescent profile (melt curve) that are then converted to melting temperature (Tm), by calculating the inflection point of the melt curve.

 

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