Protein Expression and Thermal Shift Assay

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This protocol combines cell-free protein expression, Strep-tag magnetic bead purification, and a Protein Thermal Shift Assay using SYPRO Orange into a single end-to-end workflow. S tarting from a DNA template plate, the protocol expresses protein in a CFPS reaction, purifies the tagged product via Strep-II tag, and then characterizes thermal unfolding of the purified protein by extrinsic fluorescence. The full workflow leverages our Nebula RAC system: Agilent Bravo 96 stamping of DNA into CFPS master mix, Cytomat incubation, Biotek/Agilent reagent dispense and bead binding on the Bravo Shaker, automated wash and elution on the Bravo 96, Echo acoustic transfer of SYPRO dye into assay plates, sample stamping in triplicate, Plateloc sealing, and thermal ramping on a Bio-Rad CFX Opus qPCR instrument. This method is best suited for screening and ranking protein variants by thermal stability directly from DNA, delivering consistent Tm values across a large sample set. Show more