Looking for ugly ducklings: The role of the stability of BrdU-antibody complex and the improved method of the detection of DNA replication

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61989592%3A15110%2F17%3A73580357" target="_blank" >RIV/61989592:15110/17:73580357 - isvavai.cz</a>

Result on the web

<a href="http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0174893&type=printable" target="_blank" >http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0174893&type=printable</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1371/journal.pone.0174893" target="_blank" >10.1371/journal.pone.0174893</a>

Result language

angličtina

Original language name

Looking for ugly ducklings: The role of the stability of BrdU-antibody complex and the improved method of the detection of DNA replication

Original language description

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&apos;-deoxyuridine.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

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OECD FORD branch

30101 - Human genetics

Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Publication year

2017

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

PLoS One

ISSN

1932-6203

e-ISSN

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Volume of the periodical

12

Issue of the periodical within the volume

3

Country of publishing house

US - UNITED STATES

Number of pages

18

Pages from-to

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UT code for WoS article

000399174800099

EID of the result in the Scopus database

2-s2.0-85016813268