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 gamma-H2AX. In vitro and in vivo characterization showed the specificity of the gamma-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.