Protocol abstract

Nature Protocols 2, 2285 - 2294 (2007)
Published online: 13 September 2007 | doi:10.1038/nprot.2007.320

Subject Categories: Genomics and proteomics | Imaging | Cell and developmental biology | Biochemistry and protein analysis

Fluorescence detection of protein clusters in individual cells and tissue sections by using toponome imaging system: sample preparation and measuring procedures

Manuela Friedenberger1, Marcus Bode1,2, Andreas Krusche1,2 & Walter Schubert1

This protocol details sample preparation and measurement procedures for a fluorescence technology capable of colocalizing hundreds of different proteins in a cell or tissue section. The procedure relies on fixation of samples and on the use of dye-conjugated tag libraries. To colocalize proteins, a sample is placed on the microscope stage of an imaging system (toponome imaging system (TIS)) performing sequential cycles of tag-dye incubation, imaging and bleaching to generate images for each localization cycle. TIS overcomes the spectral limitations of traditional fluorescence microscopy. Image processing reveals toponome maps, uncovering the coexistence of proteins at a location (protein clusters). The approach provides direct insight into the topological organization of proteins on a proteomic scale for the first time. If, for example, two dyes are used per cycle, 18 proteins in 4 visual fields can be colocalized in 21 h. Parallel TIS procedures using more than two dyes per cycle enhance the throughput.

  1. Molecular Pattern Recognition Research Group, Institute of Medical Neurobiology, Otto-von-Guericke-University Magdeburg, ZENIT Building, Leipziger Strasse 44, 39120 Magdeburg, Germany.
  2. ToposNomos Ltd., Belgradstrasse 55, 80796 Munich, Germany.

Correspondence to: Walter Schubert1 e-mail: