Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP
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ATPase assays are a common tool for the characterization of purified ATPases. Here, we describe a radioactive [γ-32P]-ATP-based approach, utilizing complex formation with molybdate for phase separation of the free phosphate from non-hydrolyzed, intact ATP. The high sensitivity of this assay, compared to common assays such as the Malachite green or NADH-coupled assay, enables the examination of proteins with low ATPase activity or low purification yields. This assay can be used on purified proteins for several applications including the identification of substrates, determination of the effect of mutations on ATPase activity, and testing specific ATPase inhibitors. Furthermore, the protocol outlined here can be adapted to measure the activity of reconstituted ATPases.
Originalsprog | Engelsk |
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Artikelnummer | e4676 |
Tidsskrift | Bio-protocol |
Vol/bind | 13 |
Udgave nummer | 10 |
Antal sider | 14 |
ISSN | 2331-8325 |
DOI | |
Status | Udgivet - 2023 |
Bibliografisk note
Funding Information:
This protocol was adapted from our previous work (Marek et al., 2011; Laub et al., 2017; Theorin et al., 2019; Stanchev et al., 2021). This work was funded by a grant from the Novo Nordisk Fonden (NNF18OC0034784) to T.G.P.; S.V. is a scholar of the Studienstiftung des Deutschen Volkes. Figures were created in BioRender.
Publisher Copyright:
Copyright: © 2023 The Authors; exclusive licensee Bio-protocol LLC.
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