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

Kód výsledku v 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>

Výsledek na webu

<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>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

30101 - Human genetics

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

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

Rok uplatnění

2017

Kód důvěrnosti údajů

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

Název periodika

PLoS One

ISSN

1932-6203

e-ISSN

—

Svazek periodika

12

Číslo periodika v rámci svazku

3

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

18

Strana od-do

—

Kód UT WoS článku

000399174800099

EID výsledku v databázi Scopus

2-s2.0-85016813268