Most anti-BrdU antibodies react with 2'-deoxy-5-ethynyluridine -- the method for the effective suppression of this cross-reactivity.

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  • Additional Information
    • Source:
      Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE
    • Publication Information:
      Original Publication: San Francisco, CA : Public Library of Science
    • Subject Terms:
      Microscopy, Fluorescence*; Antibodies/*immunology ; Bromodeoxyuridine/*immunology ; Deoxyuridine/*analogs & derivatives; Antibodies/chemistry ; Antibody Affinity ; Biological Transport ; Biotinylation ; Bromodeoxyuridine/chemistry ; Bromodeoxyuridine/metabolism ; Cross Reactions/immunology ; DNA/chemistry ; DNA/metabolism ; Deoxyuridine/chemistry ; Deoxyuridine/immunology ; Deoxyuridine/metabolism ; Fluorescent Dyes ; HeLa Cells ; Humans ; Staining and Labeling
    • Abstract:
      5-Bromo-2'-deoxyuridine (BrdU) and 2'-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes is used for EdU localisation. We have performed an analysis of ten samples of antibodies against BrdU with respect to their reactivity with EdU. Except for one sample all the others evinced reactivity with EdU. A high level of EdU persists in nuclear DNA even after the reaction of EdU with fluorescent azido-dyes if the common concentration of dye is used. Although a ten-time increase of azido-dye concentration resulted in a decrease of the signal provided by anti-BrdU antibodies, it also resulted in a substantial increase of the non-specific signal. We have shown that this unwanted reactivity is effectively suppressed by non-fluorescent azido molecules. In this respect, we have tested two protocols of the simultaneous localisation of incorporated BrdU and EdU. They differ in the mechanism of the revelation of incorporated BrdU for the reaction with antibodies. The first one was based on the use of hydrochloric acid, the second one on the incubation of samples with copper(I) ions. The use of hydrochloric acid resulted in a significant increase of the non-specific signal. In the case of the second method, no such effect was observed.
    • References:
      Curr Protoc Cytom. 2011 Jan;Chapter 7:Unit 7.38. (PMID: 21207361)
      J Org Chem. 2010 Dec 17;75(24):8604-14. (PMID: 21086959)
      J Biol Chem. 2004 May 7;279(19):20067-75. (PMID: 14982920)
      Nucleic Acids Res. 2010 Sep;38(16):e167. (PMID: 20627869)
      J Cell Sci. 2002 Nov 1;115(Pt 21):4037-51. (PMID: 12356909)
      Genes Dev. 2000 Nov 15;14(22):2855-68. (PMID: 11090133)
      J Med Chem. 1981 Dec;24(12):1385-8. (PMID: 6796687)
      Anal Biochem. 2011 Oct 1;417(1):112-21. (PMID: 21683679)
      J Cell Biol. 1998 Mar 23;140(6):1285-95. (PMID: 9508763)
      Proc Natl Acad Sci U S A. 2008 Feb 19;105(7):2415-20. (PMID: 18272492)
      Biophys J. 1969 Nov;9(11):1281-311. (PMID: 4982056)
      Histochem J. 1992 May;24(5):251-9. (PMID: 1376726)
      Mol Cell Biol. 2008 Sep;28(17):5265-74. (PMID: 18573882)
      PLoS One. 2012;7(12):e52584. (PMID: 23300711)
      Mol Cell Biol. 2007 Aug;27(16):5806-18. (PMID: 17515603)
    • Accession Number:
      0 (Antibodies)
      0 (Fluorescent Dyes)
      9007-49-2 (DNA)
      G34N38R2N1 (Bromodeoxyuridine)
      G373S00W2J (5-ethynyl-2'-deoxyuridine)
      W78I7AY22C (Deoxyuridine)
    • Publication Date:
      Date Created: 20121229 Date Completed: 20130610 Latest Revision: 20211021
    • Publication Date:
      20240829
    • Accession Number:
      PMC3525573
    • Accession Number:
      10.1371/journal.pone.0051679
    • Accession Number:
      23272138