nanoDSF
Differential Scanning Fluorimetry (nanoDSF)
nanoDSF monitors the intrinsic fluorescence of tryptophan and tyrosine residues as a protein is heated, detecting unfolding transitions without any added dyes. The ratio of fluorescence emission at 350 nm to 330 nm shifts as buried aromatic residues become solvent-exposed, yielding a melting temperature (Tm) (temperature where 50% of the protein is unfolded). Our instrument simultaneously measures back-reflection to detect the onset of aggregation (Tagg), which helps distinguish between partial unfolding and aggregation events.
We use Nanotemper Prometheus NT 48 that can measure upto 48 samples in a single run.
Experiment Examples
- Basic melting temperature measurement to characterize protein stability.
- Screening buffer formulations (pH/salt/additives) to find the condition that maximizes the stability (Tm) of a protein.
- Confirming that a ligand (peptide, cofactor, nucleic acid, small molecule, drug candidate) binds its target by measuring a thermal shift (ΔTm). In ideal cases, binding affinity can also be determined by monitoring the thermal shift upon titration with the ligand.
- Comparing the Tm of point mutants of a protein to characterize changes in folding or stability and/or identify a stabilizing mutation.
- Ranking the relative stability of a protein across a pH/buffer/additive series to define an optimal storage condition.
- Measuring the effect of salt concentration or a stabilizing excipient (e.g. sucrose, arginine) on aggregation onset.
- Assessing batch-to-batch consistency of a purified protein by comparing melting profiles.
Instrument
NanoTemper Prometheus NT.48
Sample Requirements
The technique uses very little material, but requirements vary with experiment type:
- Thermal stability: Typically 12-15 µL.
- Buffer screening to optimize protein stability: 12-15 µL per condition; prepare each buffer condition with identical protein concentration.
- Stabilization upon ligand/drug binding (thermal shift): Protein at fixed concentration (as above, per condition), plus ligand for titration or single saturating concentration; include a protein alone reference and a solvent-matched control if the ligand is in a specific solvent.
- Buffer constraints: Avoid additives with strong intrinsic fluorescence or high turbidity. Reducing agents, salts, and most excipients are tolerated.
- Membrane proteins: Compatible with detergents and many nanodisc/SMALP preparations; verify that the detergent does not contribute strong fluorescent background.
Practicalities
- Sample have to be spinned down at high speed for a few minutes and transfered to a fresh tube
- Ideally fill samples in PCR tubes
