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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Detalles Bibliográficos
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
Descripción
Sumario: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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