Laccase-Mediated Catalyzed Fluorescent Reporter Deposition for Live-Cell Imaging.
catalyzed reporter deposition
enzymes
fluorescence microscopy
imaging agents
laccase
Journal
Chembiochem : a European journal of chemical biology
ISSN: 1439-7633
Titre abrégé: Chembiochem
Pays: Germany
ID NLM: 100937360
Informations de publication
Date de publication:
15 01 2020
15 01 2020
Historique:
received:
24
09
2019
pubmed:
27
9
2019
medline:
6
1
2021
entrez:
27
9
2019
Statut:
ppublish
Résumé
Catalyzed reporter deposition (CARD) is a widely established method for labeling biological samples analyzed using microscopy. Horseradish peroxidase, commonly used in CARD to amplify reporter signals, requires the addition of hydrogen peroxide, which may perturb samples used in live-cell microscopy. Herein we describe an alternative method of performing CARD using a laccase enzyme, which does not require exogenous hydrogen peroxide. Laccase is an oxidative enzyme which can carry out single-electron oxidations of phenols and related compounds by reducing molecular oxygen. We demonstrate proof-of-concept for this technique through the nontargeted covalent labeling of bovine serum albumin using a fluorescently labeled ferulic acid derivative as the laccase reporter substrate. We further demonstrate the viability of this approach by performing live-cell CARD with an antibody-conjugated laccase against a surface-bound target. CARD using laccase produces an amplified fluorescence signal by labeling cells without the need for exogenous hydrogen peroxide.
Identifiants
pubmed: 31556173
doi: 10.1002/cbic.201900593
pmc: PMC7556693
mid: NIHMS1064643
doi:
Substances chimiques
Fluorescent Dyes
0
Laccase
EC 1.10.3.2
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
98-102Subventions
Organisme : NIDDK NIH HHS
ID : DP2 DK111801
Pays : United States
Informations de copyright
© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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