The photostability of the commonly used biotin-4-fluorescein probe

Anal Biochem. 2017 Aug 15:531:78-82. doi: 10.1016/j.ab.2017.05.019. Epub 2017 May 22.


Biotin-4-fluorescein (B4F) is a commonly used fluorescent probe for studying biotin-(strept)avidin interactions. During a characterization study of an anti-biotin antibody, using B4F as the probe, we noticed a discrepancy in the expected and experimentally determined number of biotin binding sites. Analytical testing showed that the biotin moiety in the probe undergoes a photosensitized oxidation to produce a mixture of biotin sulfoxides which has the potential to impact the quantitation of binding sites using this fluorescent probe.

Keywords: Biotinylation; Fluorescein; Fluorophore; Photosensitizer; Strept(avidin); Sulfoxide.

MeSH terms

  • Biotin / analogs & derivatives*
  • Biotin / chemistry
  • Fluoresceins / chemistry*
  • Oxidation-Reduction
  • Photochemical Processes*
  • Streptavidin / chemistry


  • Fluoresceins
  • biotin-4-fluorescein
  • Biotin
  • Streptavidin