Determining Biophysical Protein Stability in Lysates by a Fast Proteolysis Assay, FASTpp

David P. Minde, Madelon M. Maurice, Stefan G. D. Rüdiger

Research output: Contribution to journalArticleAcademicpeer-review


The biophysical stability is an important parameter for protein activity both in vivo and in vitro. Here we propose a method to analyse thermal melting of protein domains in lysates: Fast parallel proteolysis (FASTpp). Combining unfolding by a temperature gradient in a thermal cycler with simultaneous proteolytic cleavage of the unfolded state, we probed stability of single domains in lysates. We validated FASTpp on proteins from 10 kDa to 240 kDa and monitored stabilisation and coupled folding and binding upon interaction with small-molecule ligands. Within a total reaction time of approximately 1 min, we probed subtle stability differences of point mutations with high sensitivity and in agreement with data obtained by intrinsic protein fluorescence. We anticipate a wide range of applications of FASTpp in biomedicine and protein engineering as it requires only standard laboratory equipment. © 2012 Minde et al.
Original languageEnglish
Article numbere46147
Pages (from-to)1-9
Number of pages9
JournalPLoS One
Issue number10
Publication statusPublished - 3 Oct 2012


  • ligand
  • protein
  • article
  • assay
  • biomedicine
  • biophysics
  • cell lysate
  • fast parallel proteolysis assay
  • fluorescence analysis
  • point mutation
  • protein cleavage
  • protein degradation
  • protein domain
  • protein engineering
  • protein folding
  • protein interaction
  • protein stability
  • protein unfolding
  • reaction time
  • temperature
  • validation process


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