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Madeline O’Donoghue Life Technologies, United States of America |
Abstract
The Protein Thermal Shift Assay (TSA) is a rapid and sensitive tool for monitoring protein thermostability, aiding in the identification of optimal buffer conditions, point mutations, sequences or conformations that favour protein stability. Additionally, protein ligand interactions can readily be investigated in a high-throughput mode, to allow for rapid identification of stabilizing and destabilizing compounds.. In the example shown, TSA is used to screen for appropriate buffer conditions to maximise protein stability, in which RecA protein produced a Tm shift greater than 10oC across a range of buffer and salt conditions. In addition, TSA approaches can be used for screening protein variants that differ in amino acid sequence, in order to select for the candidate with the desired stability under the conditions tested. Using an example of Reverse Transcription Polymerases containing a range of mutations designed to increase protein thermostability, TSA data showed an appropriate increase in Tm across the protein variants under the buffer conditions tested. Using SYPRO Orange Dye and a range of experimental conditions, TSA data have been obtained across the range of Applied Biosystems Real Time PCR Systems; including the 7900 HT, 7500 Fast and StepOnePlusTM Real Time PCR Instruments. Practical examples of the scope of TSA applications across a range of proteins and buffer conditions are discussed, demonstrating the value of TSA screening, as well as the expanded utility of the Applied Biosystems Real Time PCR Instrument family.
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