Differential Scanning Fluorimetry (DSF)

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DSF at the CMI

Differential Scanning Fluorimetry (DSF) measures protein unfolding by monitory changes in fluorescence as a function of temperature. Conventional DSF uses a hydrophobic fluorescent dye that binds to proteins as they unfold. NanoDSF measures changes in intrinsic protein fluorescence as proteins unfold.

DSF Theory

The CMI has a modified Life Technologies Quant Studio 6/7, for conventional DSF.

The CMI has a Prometheus NT.Plex instrument from NanoTemper Technologies with aggregation optics. The CMI has these Data collection and analysis software packages: PR.ThermControl for thermal stability data collection, PR.ChemControl for chemical stability data collection, PR.TimeControl for time interval data collection, and PR.Stability Analysis for advanced data analysis.

 

DSF (Conventional DSF, Protein Thermal Shift Analysis)

Conventional 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.

NanoDSF

NanoDSF is a modified differential scanning fluorimetry method which monitors intrinsic tryptophan and tyrosine fluorescence as a function of temperature, time, or denaturant concentration. Tryptophan and tyrosine fluorescence intensity and wavelength maximum will vary as the local chemical environment changes, with significant changes occurring as buried or packed aromatic side chains become solvent exposed upon unfolding.  NanoDSF measures fluorescence intensity at 350 nm and 330 nm and compares the ratio as a function of temperature or denaturant concentration. NanoDSF can be used for a broader range of protein samples than traditional DSF and has significantly higher throughput and lower sample consumption than DSC or CD. Free energies of folding and temperatures of unfolding measured using NanoDSF are comparable to values determined by DSC for a range of sample types. The only significant limitation is that the protein of interest must contain aromatic amino acids (tryptophan or tyrosine).

 

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 

qPCR Resources

NanoDSF Resources

NanoDSF Supplies

  • NT.Plex Capillary Chips
    • 2x 8 Standard 24-Capillary Chips, NanoTemper Catalog # PR-AC002
    • 2x 8 High Sensitivity 24-Capillary Chips, NanoTemper Catalog # PR-AC006

  • 384-well plates for loading capillaries
  • Protein samples, ligands, buffers