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The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2 '-Deoxyuridine in Cellular DNA under Various Conditions
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SYSNO ASEP 0446574 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2 '-Deoxyuridine in Cellular DNA under Various Conditions Author(s) Ligasová, A. (CZ)
Liboska, Radek (UOCHB-X) RID, ORCID
Rosenberg, Ivan (UOCHB-X) RID, ORCID
Koberna, K. (CZ)Number of authors 4 Source Title PLoS ONE. - : Public Library of Science - ISSN 1932-6203
Roč. 10, č. 7 (2015), e0132393/1-e0132393/16Number of pages 16 s. Publication form Online - E Language eng - English Country US - United States Keywords anti-halodeoxyuridine antibodies ; affinity assay ; DNA Subject RIV CE - Biochemistry R&D Projects TA03010598 GA TA ČR - Technology Agency of the Czech Republic (TA ČR) TA03010719 GA TA ČR - Technology Agency of the Czech Republic (TA ČR) LO1304 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) NV15-31604A GA MZd - Ministry of Health (MZ) Institutional support UOCHB-X - RVO:61388963 UT WOS 000358162300128 EID SCOPUS 84941347705 DOI 10.1371/journal.pone.0132393 Annotation We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority. Workplace Institute of Organic Chemistry and Biochemistry Contact asep@uochb.cas.cz ; Kateřina Šperková, Tel.: 232 002 584 ; Jana Procházková, Tel.: 220 183 418 Year of Publishing 2016 Electronic address http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132393
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