Abstract
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.
Highlights
The most widely used alternative approach is based on the use of 5-ethynyl-2'deoxyuridine (EdU; [8])
The techniques based on based on the anchored oligonucleotides containing 5bromo-2'-deoxyuridine (BrdU) are still an important tool for cell cycle analysis and studies focused on DNA replication and chromatin organization
We have used synthetic biotinylated oligonucleotides modified with BrdU for an affinity analysis of the particular anti-bromodeoxyuridine antibody clones
Summary
5-Bromo-2'-deoxyuridine (BrdU) is commonly used for the detection of the cells in the S phase of the cell cycle [1,2,3,4]. The microinjection techniques are relatively time-consuming, require special equipment and cannot be used if a very high number of labelled cells is necessary In this respect, the techniques based on BrdU are still an important tool for cell cycle analysis and studies focused on DNA replication and chromatin organization. It is obvious that particular antibody clones differ in their ability to detect BrdU incorporated in cellular DNA under various conditions, such comparison experiments are time consuming. It arises from the high number of BrdU detection systems. Used, it does not cause a considerable loss of DNA and in combination with IdU it provides sensitivity sufficient for the analysis of the cell cycle
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