Abstract
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.
Highlights
5-Bromo-2’-deoxyuridine (BrdU) is effectively incorporated into the newly synthesised DNA by cellular DNA polymerases
The cells were incubated in a solution consisting of 1× buffer for exonuclease III, 0.1 mM CaCl2, 0.4 U/μl exonuclease III; DNase I and primary antibody raised against BrdU, if not stated otherwise
The stability of BrdU-antibody complex and the improved method of the detection of DNA replication solution composed of 1× buffer for DNase I, DNase I and the primary antibody raised against BrdU
Summary
5-Bromo-2’-deoxyuridine (BrdU) is effectively incorporated into the newly synthesised DNA by cellular DNA polymerases. The widely-used protocols for BrdU detection in DNA are based on the acid treatment where the acid concentration is usually between 1 and 4 M [2, 4, 18] Such treatment results in depurination and cleavage of the DNA making the BrdU accessible for the reaction with the specific antibodies. According to our nonpublished data, the enzymatic approach is strongly dependent on the use of the appropriate anti-BrdU antibodies as well as the fixation used. These are probably the main reasons why this method is far less used than other methods of BrdU revelation. The optimised method highly preserved the reactivity of the tested proteins with antibodies and did not exhibit a significant impact on the fluorescent proteins
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