Nano differential scanning fluorimetry

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Thermal unfolding curves and unfolding transition midpoints of two monoclonal antibodies. (A) Thermal unfolding curves of two monoclonal antibodies in the presence of 25mM Na-Citrate at different pH vlaues. Insets show the pH-dependence of the first unfolding midpoint (Tm1). (B) Dependence of Tm1 and Tm2 on the pH of the buffer of all tested conditions.

nanoDSF is a modified differential scanning fluorimetry method to determine protein stability employing intrinsic tryptophan or tyrosin fluorescence.[1][2]

Protein stability is typically addressed by thermal or chemical unfolding experiments.[3] In thermal unfolding experiments, a linear temperature ramp is applied to unfold proteins, whereas chemical unfolding experiments use chemical denaturants in increasing concentrations. The thermal stability of a protein is typically described by the 'melting temperature' or 'Tm', at which 50% of the protein population is unfolded, corresponding to the midpoint of the transition from folded to unfolded.

In contrast to conventional DSF methods, nanoDSF uses tryptophan or tyrosin fluorescence to monitor protein unfolding. Both the fluorescence intensity and the fluorescence maximum strongly depends on the close surroundings of the tryptophan.[4] Therefore, the ratio of the fluorescence intensities at 350 nm and 330 nm is suitable to detect any changes in protein structure, for example due to protein unfolding.

Its applications include antibody engineering, membrane protein research, quality control and formulation development.[5]

References[edit]

  1. ^ Käufl F, Rapp F. "nanoDSF". NanoTemper Technologies GmbH. 
  2. ^ "Protein Stability Measurement Instrument". Genetic Engineering & Biotechnology News. 15 February 2015. 
  3. ^ Senisterra G, Chau I, Vedadi M (April 2012). "Thermal denaturation assays in chemical biology". Assay and Drug Development Technologies. 10 (2): 128–36. doi:10.1089/adt.2011.0390. PMID 22066913. 
  4. ^ Lakowicz JR (2006). Principles of Fluorescence Spectroscopy (3rd ed.). Springer US. ISBN 978-0-387-31278-1. 
  5. ^ Käufl F, Rapp F. "Application Notes: nanoDSF Technology". NanoTemper Technologies GmbH. 

Further reading[edit]