Differential Scanning Fluorimetry (DSF)

Differential Scanning Fluorimetry (DSF) uses a real-time PCR instrument to monitor thermally induced protein denaturation by measuring changes in fluorescence of a dye that binds preferentially to unfolded protein (such as Sypro Orange, which binds to hydrophobic regions of proteins exposed by unfolding).  This experiment is also known as a Protein Thermal Shift Assay, because shifts in the apparent melting temperature can be measured upon the addition of stabilizing or destabilizing binding partners or buffer components.

The CMI has a modified Quant Studio 6 instrument from Life Technologies which is used primarily as a resource for differential scanning fluorimetry (protein thermal shift analysis) but may also be used for 96-well RT-PCR experiments

For information on access fees, policies and getting started at the CMI, see the CMI Access Page.

 

CMI DSF Getting Started Guide
 

Protein Thermal Shift Studies manual from Applied Biosystems by Life Technologies

All Experiments:

  • 96-well FAST-block optical plate, eg.: LifeTechnologies MicroAmp FAST optical 96-well reaction plate, 0.1 ml, 4346907
  • optical adhesive film, eg.: LifeTechnologies MicroAmp Optical Adhesive Film, 4360954

DSF/Protein Thermal Shift Experiments

  • DSF compatible dye, eg.: LifeTechnologies Protein Thermal Shift Dye Kit, 4461146 (Sypro Orange)
  • samples, ligands, buffers 

qPCR Experiments

  • qPCR reagents (eg. LifeTechnologies PowerUp SYBR Green Master Mix, A25742)
  • primers and templates 

Samples
Concentration of protein and dye should be optimized for best results

  • protein:   0.05-10 ug/well (1-5 µg)
  • Sypro Orange dyes
    • 1000X Protein Thermal Shift dye kit, final concentration 0.2X-5X (1X)
    • 5000X Sypro Orange Protein Stain, final concentration 1X-20X (5X)
  • total volume:  10-30 µl/well (20 µl), perform 3-4 replicates

 Buffers
DSF can be performed in a wide variety of buffers.  When comparing thermal stability of mutant proteins or protein/ligand complexes it is important to match buffers between samples.  Differences in buffer composition can result in variability in protein thermal stability.  In fact, DSF is a good tool for comparing the effect of different buffers on protein stability.

qPCR Resources

CMI qPCR Getting Started Guide

Quant Studio 6/7 Quick Reference Guide from Applied Biosystems by Life Technologies