Differential scanning fluorometry protocol

Thermal shift experiment to screen buffer conditions with three salt concentrations, in triplicate

Materials

• 96-well buffer screen, pH ~3-9
• SYPRO orange dye
• 96 well plate for qPCR cycler
• Protein solution at 250 µM
• 5M NaCl solution

Protocol

1. Dilute SYPRO Orange to 250X by 20-fold dilution.
2. Make three sets of buffer screens to test three different salt concentrations in 96 well transparent flat bottom plates for a total of 3 plates with 200 µL per well.
   • 50 mM buffer (add 10 µL of 1M stocks from plate per well)
   • 0, 200, 500 mM NaCl (2, 8, 20 µL from 5M stock)
   • remaining volume ddH2O
3. Set up DSF assay in nine 96 well optical PCR plates.
   • Transfer 48 µL of each buffer solution to a PCR plate.
   • Add 1 µL diluted SYPRO dye.
   • Add 1 µL protein stock to final concentration of 5 µM.
4. Run thermal melts in triplicate.

Notes

• Add SYPRO Orange right before doing the melts to avoid photobleaching.
• Acceptable protein concentration range ~2-10 µM.