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The Cramer Lab

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        Molecular Bioprocessing Research

 

      Other techniques

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Atomic Force Microscopy (AFM) 

Probing protein selectivity in hydroxyapatite using single molecule atomic force microscopy

Atomic force microscopy (AFM) is employed to directly measure the forces involved between proteins and chromatographic surfaces in order to gain insights into the principles that govern such interactions. Single molecule AFM is used to probe the energetics of protein interactions with Ceramic hydroxyapatite by functionalizing the AFM probe with the protein of interest and performing force-distance measurements. The force of interactions between the immobilized protein and the CHT surface are investigated in the presence of different mobile phase modifiers. The relative binding strengths obtained from these experiments are compared to column chromatographic retention data. By controlling the orientation of the protein binding to a surface, fundamental insights can be obtained regarding the binding energies associated with these interactions. AFM can indeed be employed to examine protein chromatographic surface interactions, opening new possibilities for examining preferred binding orientations in protein chromatography.

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Fig 1. Representation of the chemistry employed for tethering the protein to the AFM tip

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Fig 2. Force distance cycle of protein interacting with a CHT surface. A) Approach towards surface B) tip-surface contact C) retract away from surface

 

 

Quartz Crystal Microbalance (QCM) 

Investigation of kinetics of protein binding

The kinetics of protein binding to chromatographic ligands in the presence of different mobile phase modifiers need to be understood at a fundamental level. This requires detailed understanding of the structural characteristics of these mobile phase modifiers which give rise to unique selectivities in protein separation. Quartz crystal microbalance (QCM) is being employed to obtain quantitative information about the kinetics of desorption of proteins from self-assembled monolayers (SAM) of chromatographic ligands. The QCM-D is capable of measuring structural, viscoelastic properties and mass changes in real time with nano-sensitivity. A SAM surface of a particular ligand of interest is created which mimics the ligand on a resin surface. Hence binding of protein to the surface can be evaluated in a flow through manner which mimics the situation in a column setting.

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Fig.3 Protein desorption behavior in the presence of A) Spermine B) Spermidine and C) bis(hexamethylene)triamine

 

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