A protocol for visualizing active cathepsin K in osteoclasts with a quenched-fluorescence-activity-based probe

Herein, we provide a protocol for visualizing active osteoclast cathepsin K (CatK) with the quenched-fluorescent-activity-based probe qTJK17. We describe steps for isolating peripheral blood mononuclear cells, their differentiation into osteoclasts, and TRAP staining using an acid phosphatase leukoc...

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Autores principales: Korba, Aleksandra, Ciastoń, Izabela, Kozieł, Joanna, Kasperkiewicz, Paulina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10374869/
https://www.ncbi.nlm.nih.gov/pubmed/37481728
http://dx.doi.org/10.1016/j.xpro.2023.102465
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author Korba, Aleksandra
Ciastoń, Izabela
Kozieł, Joanna
Kasperkiewicz, Paulina
author_facet Korba, Aleksandra
Ciastoń, Izabela
Kozieł, Joanna
Kasperkiewicz, Paulina
author_sort Korba, Aleksandra
collection PubMed
description Herein, we provide a protocol for visualizing active osteoclast cathepsin K (CatK) with the quenched-fluorescent-activity-based probe qTJK17. We describe steps for isolating peripheral blood mononuclear cells, their differentiation into osteoclasts, and TRAP staining using an acid phosphatase leukocyte kit. We then detail visualization of active CatK. The probe qTJK17 includes a reactive group, acyloxymethylketone, that binds to the CatK active site, recognition sequence, and fluorescence donor-acceptor pair. This protocol can determine the exact localization of active CatK in osteoclasts. For complete details on the use and execution of this protocol, please refer to Janiszewski et al. (2023).(1)
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spelling pubmed-103748692023-07-29 A protocol for visualizing active cathepsin K in osteoclasts with a quenched-fluorescence-activity-based probe Korba, Aleksandra Ciastoń, Izabela Kozieł, Joanna Kasperkiewicz, Paulina STAR Protoc Protocol Herein, we provide a protocol for visualizing active osteoclast cathepsin K (CatK) with the quenched-fluorescent-activity-based probe qTJK17. We describe steps for isolating peripheral blood mononuclear cells, their differentiation into osteoclasts, and TRAP staining using an acid phosphatase leukocyte kit. We then detail visualization of active CatK. The probe qTJK17 includes a reactive group, acyloxymethylketone, that binds to the CatK active site, recognition sequence, and fluorescence donor-acceptor pair. This protocol can determine the exact localization of active CatK in osteoclasts. For complete details on the use and execution of this protocol, please refer to Janiszewski et al. (2023).(1) Elsevier 2023-07-22 /pmc/articles/PMC10374869/ /pubmed/37481728 http://dx.doi.org/10.1016/j.xpro.2023.102465 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Korba, Aleksandra
Ciastoń, Izabela
Kozieł, Joanna
Kasperkiewicz, Paulina
A protocol for visualizing active cathepsin K in osteoclasts with a quenched-fluorescence-activity-based probe
title A protocol for visualizing active cathepsin K in osteoclasts with a quenched-fluorescence-activity-based probe
title_full A protocol for visualizing active cathepsin K in osteoclasts with a quenched-fluorescence-activity-based probe
title_fullStr A protocol for visualizing active cathepsin K in osteoclasts with a quenched-fluorescence-activity-based probe
title_full_unstemmed A protocol for visualizing active cathepsin K in osteoclasts with a quenched-fluorescence-activity-based probe
title_short A protocol for visualizing active cathepsin K in osteoclasts with a quenched-fluorescence-activity-based probe
title_sort protocol for visualizing active cathepsin k in osteoclasts with a quenched-fluorescence-activity-based probe
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10374869/
https://www.ncbi.nlm.nih.gov/pubmed/37481728
http://dx.doi.org/10.1016/j.xpro.2023.102465
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