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Case Study on Live Cell Apoptosis-Assay Using Lamin-Chromobody Cell-Lines for High-Content Analysis

  • Kourosh Zolghadr
  • Jacqueline Gregor
  • Heinrich Leonhardt
  • Ulrich RothbauerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 911)

Abstract

The understanding of cellular processes and their physiopathological alterations requires comprehensive data on the abundance, distribution, modification and interaction of cellular components. On the one hand, artificially introduced fluorescent fusion proteins provide information about their distribution and dynamics in living cells but not on endogenous factors. On the other hand, antibodies can detect endogenous proteins, posttranslational modifications and other cellular components but mostly in fixed and permeabilized cells. Here we highlight a new technology based on the antigen-binding domain of heavy-chain antibodies (VHH) from Camelidae. We have demonstrated that these VHH domains can be fused with fluorescent proteins and expressed in living cells. Those fluorescent antigen-binding proteins-called chromobodies-can be used to detect and trace proteins and other cellular components in vivo. In principle chromobodies can detect any antigenic structure including posttranslational modifications or nonprotein components and thereby dramatically expand the quality and quantity of information that can be gathered in high-content analyses. Here we demonstrate the suitability of this technology to follow apoptosis in living cells in real time.

Key words

Antibodies Chromobodies High-content analysis GFP Fluorescent proteins Live cell microscopy Apoptosis HCA assay 

Notes

Acknowledgements

The authors declare competing financial interests. This work was supported by the GO-Bio Program of the BMBF (Federal Ministry of Science, Germany).

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Kourosh Zolghadr
    • 1
    • 2
  • Jacqueline Gregor
    • 2
  • Heinrich Leonhardt
    • 3
  • Ulrich Rothbauer
    • 1
    • 4
    Email author
  1. 1.Natural and Medical Science Institute at the University of TuebingenUniversity of TuebingenReutlingenGermany
  2. 2.ChromoTek GmbHMartinsriedGermany
  3. 3.CIPS, Center for Integrated Protein Science at the Department of Biology IILudwig Maximilians University MunichPlanegg-MartinsriedGermany
  4. 4.ChromoTek GmbHPlanegg-MartinsriedGermany

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