Experimental techniques

  • Recombinant protein expression in E. coli, yeast and mammalian cells

  • Screening of protein expression using fluorescence-detection size exclusion chromatography (FSEC)

  • Protein purification using affinity tag-, ion exchange- and size exclusion-chromatography

  • Analysis of protein function

    o   Radioactive 86Rb+ flux assay

    o   Single channel recordings in horizontal lipid bilayers

    o   Patch-clamp electrophysiology

    o   Fluorescence stopped flow flux assay (SX20 sequential mixing stopped flow, Applied Photophysics)

    o   access to fluorescence spectrophotometer and ITC

    o   access to automated crystallography equipment to setup up conventional screens and LCP screens

    o   access to equipment to prepare cryo-EM grids

    o   access to 300 kV Titan Krios electron microscopes at the New York Structural Biology Center (NYSBC)

 
Picture1.png

Feature: Protein expression

Following standard molecular biology techniques for cloning and engineering genes of interest, we express ion channel proteins and accessory proteins in E. coli, yeast, insect cells, or mammalian expression systems.

We perform state-of-the-art protein purification with AKTA explorer systems at different temperatures. Optimized for the specific needs of each protein we reconstitute channels into liposomes or lipid nanodiscs for functional and structural analyses.

Picture1.png

Feature: protein function

We are equipped for a wide range of electrophysiological experiments ranging from single channel recordings in planar lipid bilayers (including MiniOrbit), two-electrode voltage clamp recordings, to patch-clamp experiments (including Port-a-patch). Furthermore, we use a fluorescence-based sequential-mixing stopped-flow assay to analyze ion channel kinetics.

We complement our studies by long-standing collaborations in the fields of high-speed AFM, MD-simulations, and NMR.

Picture1.png

Feature: structural biology

We assess the quality of our protein samples using in-house negative stain EM. Optimized samples are then used for X-ray crystallography or to prepare grids for single particle cryoEM using an in-house Vitrobot. Also available in-house is a Glacios cryo-TEM for grid screening and data collection.

High-resolution data collection is also available at the New York Structural Biology Center (NYSBC) or other national centers.