Open AccessGeneration of an alpaca‐derived nanobody recognizing γ‐H2AX
Author(s) -
Rajan Malini,
Mortusewicz Oliver,
Rothbauer Ulrich,
Hastert Florian D.,
Schmidthals Katrin,
Rapp Alexander,
Leonhardt Heinrich,
Cardoso M. Cristina
Publication year - 2015
Publication title -
febs open bio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.718
H-Index - 31
ISSN - 2211-5463
DOI - 10.1016/j.fob.2015.09.005
Subject(s) - epitope , transfection , antibody , dna , in vitro , single domain antibody , computational biology , microbiology and biotechnology , biology , epitope mapping , antigen , chemistry , gene , genetics
Post‐translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single‐chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ‐H2AX. In vitro and in vivo characterization showed the specificity of the γ‐H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.