Differential Scanning Fluorimetry (DSF)

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

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 the Life Technologies Protein Thermal Shift Analysis software for DSF data fitting.

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).

 

Data Files - About CMI Data Files

Users are responsible for storage of all raw and processed data collected at the CMI.

  • Users should have a plan to copy or transfer all raw and process data to their own local or cloud storage system.
  • While the CMI allows temporary local storage of CMI User data on the instrument computer, we make no guarantees on the security or long-term availability of any data at the CMI.
  • For most (but not all) CMI technologies, the raw data files and recommended readable exports are relatively small and can be readily transferred electronically. 
  • See specific instruments for exceptions and for details about the software, data file types and recommended data exports. 

Data Sharing:

  • Currently, a Generalist Repository is the recommended data repository for most CMI data types, as stable specialist data repositories have not been established.

Data Files - DSF - QuantStudio 6/7

Technology Differential Scanning Fluorimetry (DSF)
Instrument Life Technologies Quant Studio 6/7
Recommended Repository Generalist Repository    
       
Software Type Data Collection
Current Version QS Real-Time PCR Software, version 1.7.1
Data Files (Type, ~size) experiment file .eds 2-10 MB/plate
       
Software Type Data Analysis
Current Version Applied Biosystems Protein Thermal Shift, version 1.2
Data Files (Type, ~size) experiment file .eds 2-10 MB/plate
Readable Exports raw data .csv 2 MB/plate
  analyzed data .csv 12 KB/project
  analyzed data .txt 29 KB/project

Data Files - DSF - Prometheus

Technology Nano Differential Scanning Fluorimetry (nanoDSF)
Instrument NanoTemper Prometheus NT.Plex
Recommended Repository Generalist Repository
       
Software Type Data Collection (Thermal Denaturation)
Current Version PR.ThermControl, Version 2.3.1
Data Files (Type, ~size) experiment file .prc 10-30 MB/project
  raw data .xslx 2 MB/project
       
Software Type Data Analysis
Current Version PR.Stability Analysis, Version 1.1
Data Files (Type, ~size) analysis file .pra 2-6 MB/project
Readable Exports processed data .xslx ~500 KB/sample
  results table .xslx ~30 KB/project
       
Software Type Data Collection (Chemical Denaturation)
Current Version PR.ChemControl, Version 1.4.3
Data Files (Type, ~size) experiment file .prcc 10 MB/project
Readable Exports raw data .xslx 6 KB/sample
       
Software Type Data Collection (Time Control)
Current Version PR.TimeControl, Version 1.0.2
Data Files (Type, ~size) experiment file .prtime 2 MB/project
Readable Exports raw data .xslx 60 KB/project

DSF Data Collection Services

DSF Service Overview

In addition to instrument training, the CMI is now offering basic protein Differential Scanning Fluorimetry services, including protein thermal shift analysis and buffer optimization.

Differential Scanning Fluorimetry (DSF) with fluorescent dye or using intrinsic protein fluorescence

Life Technologies Quant Studio 6/7

  • Conventional DSF, protein thermal stability using hydrophobic dye Sypro Orange
  • Buffer optimization

NanoTemper Technologies Prometheus NT.Plex

  • NanoDSF, protein thermal stability using intrinsic protein fluorescence
  • Chemical denaturation
  • Buffer optimization

Data Collection Fees Summary

Data Collection

  • Limited Data Collection Services are offered.
  • Service fees are based on labor and supplies costs, and will be charged for all completed services, regardless of experimental outcome. 
  • Before submitting samples for data collection, users must approve the estimated charges and be given a date and time for sample delivery.
    • External Users will also be required to submit a PO and a signed CMI User Agreement.
  • Most CMI Data Collection Services include a setup fee plus a per-sample data collection fee.
    • Some services include replicate measurements by default in the per-sample fee. For others, there is a reduced-price replicate measurement fee, if collected in the same dataset.
  • Nanobody services not available to commercial users at this time.
  • Current Harvard Life Lab commercial users are offered a 25% discount off the standard commercial rates.

All Data Collection Fees

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