Open AccessLooking for ugly ducklings: The role of the stability of BrdU-antibody complex and the improved method of the detection of DNA replication
Author(s) -
Anna Ligasová,
Petr Konečný,
Ivo Frydrych,
Karel Koberna
Publication year - 2017
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0174893
Subject(s) - antibody , deoxyuridine , immunostaining , flow cytometry , primary and secondary antibodies , microbiology and biotechnology , dna , bromodeoxyuridine , biology , dna replication , fluorescence , chemistry , virology , immunohistochemistry , biochemistry , immunology , physics , quantum mechanics
5-Bromo-2′-deoxyuridine (BrdU) labelling and immunostaining is commonly used for the detection of DNA replication using specific antibodies. Previously, we found that these antibodies significantly differ in their affinity to BrdU. Our present data showed that one of the reasons for the differences in the replication signal is the speed of antibody dissociation. Whereas highly efficient antibodies created stable complexes with BrdU, the low efficiency antibodies were unstable. A substantial loss of the signal occurred within several minutes. The increase of the complex stability can be achieved by i ) formaldehyde fixation or ii ) a quick reaction with a secondary antibody. These steps allowed the same or even higher signal/background ratio to be reached as in the highly efficient antibodies. Based on our findings, we optimised an approach for the fully enzymatic detection of BrdU enabling the fast detection of replicational activity without a significant effect on the tested proteins or the fluorescence of the fluorescent proteins. The method was successfully applied for image and flow cytometry. The speed of the method is comparable to the approach based on 5-ethynyl-2′-deoxyuridine. Moreover, in the case of short labelling pulses, the optimised method is even more sensitive. The approach is also applicable for the detection of 5-trifluoromethyl-2'-deoxyuridine.