Protein Thermal Shift Software & Reagents
Protein Thermal Shift™ Applications
Protein Stability Screening:
High-Throughput Ligand Screening:
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™ Assay
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.
- User Guide: Protein Thermal Shift™ Studies
- Application Note: Protein Thermal Shift™ Studies
- Product Bulletin: Protein Thermal Shift™ Studies
- Protein Thermal Shift™ Data Sheet
- Protein Thermal Shift™ Studies Buffer Screening Quick Reference
- Protein Thermal Shift™ Studies Mutation Screening Quick Reference
- Protein Thermal Shift™ Studies Ligand Screening Quick Reference
- Poster: Protein Thermal Shift Assay is an Excellent High Throughput Screening Strategy To Identify Optimal Buffer Conditions And Ligands Required For Maximizing YraM Protein Crystallization Success