nano Differential Scanning Fluorimetry | nanoDSF

Overview

NanoDSF is a novel, label-free method for the analysis of thermal and chemical protein unfolding. It allows for quantifying the melting temperature of a protein. In addition, nanoDSF can determine the colloidal stability (aggregation onset temperature) by back-reflection optics.

Intrinsic tryptophan fluorescence of proteins is strongly dependent on their 3D-structure and hence the local surroundings of the tryptophan residues. Using chemical denaturants or a thermal gradient, proteins can be unfolded, which leads to changes in their intrinsic tryptophan fluorescence. This translates into fluorescence emission peak shifts and intensity changes. NanoDSF monitors these fluorescence changes with high resolution and can reveal even multiple unfolding transitions. NanoDSF is therefore highly applicable in antibody engineering, membrane protein characterization and protein quality control.

The figure illustrates the principle behind thermal protein unfolding: Increasing temperature causes tryptophan residues becoming solvent exposed upon unfolding of the three-dimensional protein structure. NanoDSF monitors the concurrent changes in tryptophan fluorenscence at 330 and 350 nm wavelength.

In order to detect protein aggregation, the Prometheus NT.48 nanoDSF device features also back-reflection optics. Normally, visible light passes through the capillaries containing the protein sample of interest without any interference and is reflected by a mirror on the capillary tray and then quantified by the detector. If the protein sample contains aggregated particles, the incident light is scattered by these particles. The loss of reflected intensity is a precise measure for protein aggregation.

Technology

NanoDSF is a differential scanning fluorimetry method able to analyze the conformational stability and colloidal stability (aggregation behavior) of proteins under different thermal and chemical conditions.

The conformational stability of a protein is described by its unfolding transition midpoint Tm (°C), which is the point at which half of the protein is unfolded. The truly labelfree nanoDSF technique monitors the intrinsic tryptophan fluorescence of proteins, which is highly sensitive for the close surroundings of the tryptophan residues and which changes upon thermal unfolding.

Up to 48 capillaries are filled with 10 µl of protein sample and simultaneously scanned at 330/350 nm wavelengths. Melting temperatures are recorded by the intrinsic tryptophan fluorescence and aggregation onset temperatures are detected via back-reflection light scattering. Within 75 min, the samples are heated from 25°C to 95°C. Importantly, samples can be studied without the use of a dye and with free choice of buffer and detergent. Melting temperatures of proteins with a concentration between 5 µg/ml and 250 mg/ml can be analyzed. In order to obtain high quality aggregation onset temperatures, protein solutions commonly with concentrations above 1 mg/ml are required.

NanoDSF is a rapid and accurate method for studying the conformational and colloidal stablity of proteins and is thus perfectly suited for protein engineering, formulation development, and screening approaches.

Typical applications

  • Screenings of buffers, formulation, and buffer additives
  • Screening of detergent influence
  • Long-term protein and antibody storage optimization
  • Forced-degradation stability testing
  • Comparison of biosimilar proteins and antibodies with respect to stability and aggregation
  • Batch-to-batch comparison assays
  • Deep feature analysis (influence of mutations, modifications, conjugations on protein stability and aggregation)

Advantages

  • Low sample consumption (Only 10 µL of sample required)
  • Free choice of assay buffers (Also biological liquids possible such as serum or cell lysate and other additives/detergents)
  • Very short analysis time (enables high throughput)
  • Optimal data quality and resolution (Dual 350/330 nm UV-detection)
  • Wide temperature range (Analysis possible from 15°C to 95°C)
  • No labeling required (Close-to-native analysis possible)
  • Wide concentration range (5 µ/ml to 200 mg/ml)
  • Wide molecule size range (From 1 kDa to 1 MDa)

FAQ – General

What are typical applications for the nanoDSF Technology?

With the nanoDSF we can offer the technology of choice for easy, rapid and accurate analysis of protein folding and stability. The broad concentration range allows us to investigate biopharmaceuticals at very high concentrations e.g. used in formulation. Furthermore our technology is especially suited for applications in antibody engineering since the ultra-high resolution allows to detect and to analyse multiple transitions and unfolding events. NanoDSF also provides the possibility to measure the stability of membrane proteins in detergents since this method is truly label-free and does not require any fluorescent dye.

How many tryptophans do I need for detection?

One tryptophan is typically sufficient in your measurement due to the high sensitivity of our detector. Also tyrosin fluorescence can be detected and analysed.

How many samples can be measured in parallel?

We can measure 48 capillaries with the Prometheus NT.48.

Can I detect wavelength shifts due to unfolding?

Since the Prometheus NT.48 detects two different wavelengths, 330 and 350 nm, blue- and redshift of the tryptophan fluorescence can be distinguished.

Do I need fluorescent dyes?

No, our instruments work truly label-free and are able to detect tryptophan and tyrosin fluorescence at 330 nm and 350 nm.

How many data points can be recorded?

Due to ultra-high resolution of the nanoDSF technology you will obtain 36000 data points for an experiment with 48 samples and a heating rate of 1°C/min between 20°C and 95°C.

Can I analyse the unfolding of individual domains in my protein?

The ultra-high resolution of nanoDSF allows us to detect and to analyse multiple transitions and unfolding events.

How long does one measurement take?

For chemical unfolding, one measurement only takes 10 seconds. For thermal denaturation, it mainly depends on your heating rate. With a typical experimental setting including a heating rate of 1°C/minute and a temperature range from 20°C to 95°C, the measurement takes 75 min (without sample preparation).

FAQ – Samples

How much sample is required?

Only 10 µl of your sample is needed.

What concentration range of my samples can be measured?

2bind is able to analyse a broad concentration range from 5 µg/ml to more than 200 mg/ml.

Is it possible to analyze membrane proteins with nanoDSF?

Yes! 2bind is able to measure the stability of membrane proteins in detergents by nanoDSF since this method is truly label-free and does not require any fluorescent dye which could interfere with the detergent.

Are there any size limitations of my proteins?

No, we can also measure Mega-Dalton (MDa) protein complexes.

FAQ – Assay Conditions

What is the temperature range for the measurement?

The temperature range reaches from 15°C to 95°C with heating rates from 0.1 to 5°C/minute.

Are there any limitations in buffers or additives?

You can use any buffer and any additives, including detergents. Even measurements with autofluorescent components and biological liquids are possible to a certain extent.
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