Visualization and Quantification of Topoisomerase–DNA Covalent Complexes Using the Trapped in Agarose Immunostaining (TARDIS) Assay

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1703)

Abstract

The TARDIS assay was originally developed as a means of detecting and quantifying melphalan and cisplatin DNA adducts at the single cell level, but it has since been adapted to quantify topoisomerase DNA complexes that result from the actions of topoisomerase poisons and this is currently the main use of the assay. The method employs sensitive immunofluorescent detection to quantify topoisomerase molecules covalently coupled to DNA in what are often referred to as cleavage complexes. Free topoisomerase molecules, and other cellular constituents are first removed using salt-detergent extraction of agarose-embedded, unfixed cells. Using these stringent extraction conditions, genomic DNA remains in place in the agarose as “nuclear ghosts,” and any covalent attached molecules can be detected and quantified by immunofluorescence with a low background.

Key words

Topoisomerase Covalent complex TARDIS Immunostaining 

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Institute for Cell and Molecular BiosciencesMedical School, Newcastle UniversityNewcastle upon TyneUK

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