Poster Presentation 2014 International Biophysics Congress

Microfluidic isolation of protein complexes from cell lysates for visual analysis by quantitative electron microscopy (#276)

Dominic Giss 1 , Venkata Dandey 1 , Henning Stahlberg 1 , Thomas Braun 1
  1. Center for Cellular Imaging and Nano Analytics, Biozentrum, University of Basel, Basel, Switzerland

The investigation of protein complexes in terms of their architecture, conformation, structure and temporal persistence is a key element of systems biology. However, the dynamic and stochastic nature of the interactions involved leads to a heterogeneous mix of protein assemblies. This necessitates the quantitative analysis of such protein complexes at the single molecule level. To date, the instrumentation for the investigation of these transiently formed assemblies is still poorly developed, particularly their isolation and subsequent characterization.

Here we present a fast and simple microfluidic method for the quantitative isolation of endogenous levels of protein complexes from small amounts of cell lysates under close to physiological conditions and their subsequent visual analysis at the single molecule level by transmission electron microscopy (TEM). To this end, we employ antibodies that are conjugated via a photocleavable linker to magnetic beads being trapped in microcapillaries to bind the target proteins. Subsequently, proteins are released by photocleavage and analyzed by quantitative TEM at the single molecule level [1]. As an example, we demonstrated the isolation of endogenous levels of 26S proteasomes from cell lysates and performed a single particle analysis of the recovered holocomplexes and their intermediates.

In future, the proposed method will be combined with novel sample preparation techniques allowing the processing of single cell extracts [2] and lossless deposition of isolated structures to TEM sample carriers [3]. Ultimately, this will offer a completely new way to structurally and quantitatively characterize the dynamic and heterogeneous interaction networks of individual cells.

  1. [1] Giss D, Kemmerling S, Dandey V, Stahlberg H, Braun T (2014) Exploring the interactome: microfluidic isolation of proteins and interacting partners for quantitative electron microscopy. Anal Chem, DOI: 10.1021/ac4027803
  2. [2] Kemmerling S, Arnold SA, Bircher BA, Sauter N, Escobedo C, Dernick G, Hierlemann A, Stahlberg H, Braun T (2013) Single-cell lysis for visual analysis by electron microscopy. J Struct Biol 183: 467-473
  3. [3] Kemmerling S, Ziegler J, Schweighauser G, Arnold SA, Giss D, Muller SA, Ringler P, Goldie KN, Goedecke N, Hierlemann A, Stahlberg H, Engel A, Braun T (2012) Connecting mu-fluidics to electron microscopy. J Struct Biol 177: 128-134