Differential Scanning Fluorimetry Reaches New Peaks on the NISTmAb
molecule in the development of novel technology for therapeutic protein characterisation. In this
application note, we use the SUPR-DSF to acquire differential scanning fluorimetry (DSF) data on
the NISTmAb and compare the results against differential scanning calorimetry (DSC).
The SUPR-DSF resolved all three domains of the NISTmAb: CH2 (69°C), CH3 (83°C), and the
unusually high melting temperature of the Fab domain (94°C).[ 1] In addition, the apparent
melting temperatures agreed exceptionally well with the literature results. These results validate
you can easily obtain high quality protein stability information with the SUPR-DSF.
Results
The fluorescence spectra from the SUPR-DSF thermal melt experiment were used to calculate the
barycentric mean (BCM) to quantify the wavelength shifts of the NISTmAb as it transitioned from
the folded to the unfolded state. The NISTmAb BCM melt curve shown in Figure 1(a) illustrates
three distinct transitions: CH2 (69°C), CH3 (83°C), and Fab (94°C) domains. The SUPR-DSF can
accurately measure the unusually high Fab melting temperature due to its ability to ramp samples
up to 105°C, which is not easily possible by alternative DSF platforms. This is evident in Figure 1(a)
where the alternative DSF platform is unable to completely resolve the Fab transition.[2]
The SUPR-DSF BCM curve was processed with a first derivative and compared to the DSC data by
normalising to the Fab domain.[1] The result, shown in Figure 2(b), clearly demonstrates excellent
agreement between the two measurements depicted by all three peaks aligning with each other.
The excellent correlation between the techniques shows the intrinsic protein fluorescence
provides an orthogonal approach for measuring protein stability. This opens new possibilities by
being able to obtain high quality melting curves on 384 samples in under 2 hours with the
SUPR-DSF. The melting temperatures calculated by both methods are shown in Table 1.
Methodology
NISTmAb was prepared at 1 mg⁄mL in L-Histidine pH 6.0 buffer and dispensed in a 384-well PCR
microplate at a well volume of 10 µL. After dispensing, the plate was centrifuged for 60 seconds
and sealed with an optically clear adhesive film. The SUPR-DSF was set up to measure the
fluorescence spectra from 15°C to 105°C with a 1 °C per minute ramp rate.
Fluorescence spectra were processed with BCM calculation to quantify the spectral shift. The BCM
data was analysed as a function of temperature and processed with a first derivative. Melting
temperatures were calculated from the peaks of the derivative. Methods and results for all samples
are available on request.
Conclusion
The SUPR-DSF from Protein Stable can quickly and accurately measure melting temperatures by
monitoring the wavelength shifts of intrinsic protein fluorescence. Results shown here have
demonstrated the SUPR-DSF can obtain high quality, accurate melting temperatures of the
NISTmAb, including the unusually high melting temperature of the Fab domain (94°C). This
validates the intrinsic fluorescence based DSF method of the SUPR-DSF can achieve the same
results as DSC while increasing throughput, convenience, and lower sample consumption by
taking advantage of 384-well microplates.
