Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

Lydia Kisley, Jixin Chen, Andrea P. Mansur, Bo Shuang, Katerina Kourentzi, Mohan Vivekanandan Poongavanam, Wen Hsiang Chen, Sagar Dhamane, Richard C. Willson, Christy F. Landes

Research output: Contribution to journalArticlepeer-review

68 Scopus citations


Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by asmuch as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

Original languageEnglish (US)
Pages (from-to)2075-2080
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number6
StatePublished - Feb 11 2014


  • Bioseparations
  • Ion-exchange chromatography
  • Optical nanoscopy
  • Single-molecule kinetics

ASJC Scopus subject areas

  • General


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