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. The most common method for measuring protein thermal shifts is differential scanning fluorimetry (DSF) or thermofluor, which utilizes specialized fluorogenic dyes.
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| - Thermal Shift Assay (de)
- Thermal shift assay (en)
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| - Ein thermal shift assay (TSA, synonym Thermofluor Assay oder Differential Scanning Fluorimetry, DSF) ist eine biochemische Methode zur Messung der Änderung der Thermostabilität der Faltung eines Proteins nach Zugabe eines Bindungspartners. Sie ist dadurch eine Methode zur Bestimmung von Protein-Protein-, Protein-DNA- und Protein-Lipid-Interaktionen sowie Interaktionen zwischen Proteinen und niedermolekularen Verbindungen. (de)
- 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. The most common method for measuring protein thermal shifts is differential scanning fluorimetry (DSF) or thermofluor, which utilizes specialized fluorogenic dyes. (en)
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| - Ein thermal shift assay (TSA, synonym Thermofluor Assay oder Differential Scanning Fluorimetry, DSF) ist eine biochemische Methode zur Messung der Änderung der Thermostabilität der Faltung eines Proteins nach Zugabe eines Bindungspartners. Sie ist dadurch eine Methode zur Bestimmung von Protein-Protein-, Protein-DNA- und Protein-Lipid-Interaktionen sowie Interaktionen zwischen Proteinen und niedermolekularen Verbindungen. (de)
- 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. The most common method for measuring protein thermal shifts is differential scanning fluorimetry (DSF) or thermofluor, which utilizes specialized fluorogenic dyes. The binding of low molecular weight ligands can increase the thermal stability of a protein, as described by Daniel Koshland (1958) and Kaj Ulrik Linderstrøm-Lang and Schellman (1959). Almost half of enzymes require a metal ion co-factor. Thermostable proteins are often more useful than their non-thermostable counterparts, e.g., DNA polymerase in the polymerase chain reaction, so protein engineering often includes addingmutations to increase thermal stability. Protein crystallization is more successful for proteins with a higher melting point and adding buffer components that stabilize proteins improve the likelihood of protein crystals forming.If examining pH then the possible effects of the buffer molecule on thermal stability should be taken into account along with the fact that pKa of each buffer molecule changes uniquely with temperature. Additionally, any time a charged species is examined the effects of the counterion should be accounted for. Thermal stability of proteins has traditionally been investigated using biochemical assays, circular dichroism, or differential scanning calorimetry. Biochemical assays require a catalytic activity of the protein in question as well as a specific assay. Circular dichroism and differential scanning calorimetry both consume large amounts of protein and are low-throughput methods. The thermofluor assay was the first high-throughput thermal shift assay and its utility and limitations has spurred the invention of a plethora of alternate methods. Each method has its strengths and weaknesses but they all struggle with intrinsically disordered proteins without any clearly defined tertiary structure as the essence of a thermal shift assay is measuring the temperature at which a protein goes from well-defined structure to disorder. (en)
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