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Monoclon Antib Immunodiagn Immunother. 2019 Feb;38(1):25-29. doi: 10.1089/mab.2018.0046. Epub 2019 Feb 13.

Photobleaching Comparison of R-Phycoerythrin-Streptavidin and Streptavidin-Alexa Fluor 568 in a Breast Cancer Cell Line.

Author information

1
1 Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
2
2 Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
3
3 Department of Biology, Tonekabon Branch, Islamic Azad University, Tonekabon, Mazandaran, Iran.
4
4 ACECR, Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran, Iran.

Abstract

Fluorescent dyes are excited by light and emit light at a longer wavelength. Photobleaching is one the most important obstacles in fluorescent image capturing. Photochemical alteration of a fluorescent dye caused by several excitation/emission cycles results in a fluorophore to be unable to emit light. In this study, R-phycoerythrin (R-PE) and Alexa Fluor 568 were separately conjugated to streptavidin. The efficiency of conjugations, R-PE-streptavidin and streptavidin-Alexa Fluor 568, were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and spectrophotometry, respectively. Herceptin, a humanized therapeutic antibody, was subsequently biotinylated. The reactivity of biotin-labeled Herceptin was examined by enzyme-linked immunosorbent assay. The photobleaching of R-PE-streptavidin and streptavidin-Alexa Fluor 568 were then compared in an immunofluorescent staining on a breast cancer cell line, BT-474. Our data showed that streptavidin-Alexa Fluor 568 was more photostable than R-PE-streptavidin, which provides more time for longer viewing of labeled proteins and image capturing.

KEYWORDS:

Alexa Fluor 568; R-phycoerythrin; conjugation; photobleaching

PMID:
30759058
DOI:
10.1089/mab.2018.0046
[Indexed for MEDLINE]

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