Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP

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Standard

Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP. / Veit, Sarina; Pomorski, Thomas Günther.

In: Bio-protocol, Vol. 13, No. 10, e4676, 2023.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Veit, S & Pomorski, TG 2023, 'Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP', Bio-protocol, vol. 13, no. 10, e4676. https://doi.org/10.21769/BioProtoc.4676

APA

Veit, S., & Pomorski, T. G. (2023). Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP. Bio-protocol, 13(10), [e4676]. https://doi.org/10.21769/BioProtoc.4676

Vancouver

Veit S, Pomorski TG. Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP. Bio-protocol. 2023;13(10). e4676. https://doi.org/10.21769/BioProtoc.4676

Author

Veit, Sarina ; Pomorski, Thomas Günther. / Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP. In: Bio-protocol. 2023 ; Vol. 13, No. 10.

Bibtex

@article{e1eb1d44042946c8ba56f4e7629d35ec,
title = "Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP",
abstract = "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.",
keywords = "P-ATP, ABC transporter, ATP hydrolysis, ATPase, P-type ATPase, Radioactive assay",
author = "Sarina Veit and Pomorski, {Thomas G{\"u}nther}",
note = "Publisher Copyright: Copyright: {\textcopyright} 2023 The Authors; exclusive licensee Bio-protocol LLC.",
year = "2023",
doi = "10.21769/BioProtoc.4676",
language = "English",
volume = "13",
journal = "Bio-protocol",
issn = "2331-8325",
publisher = "bio-protocol",
number = "10",

}

RIS

TY - JOUR

T1 - Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP

AU - Veit, Sarina

AU - Pomorski, Thomas Günther

N1 - Publisher Copyright: Copyright: © 2023 The Authors; exclusive licensee Bio-protocol LLC.

PY - 2023

Y1 - 2023

N2 - 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.

AB - 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.

KW - P-ATP

KW - ABC transporter

KW - ATP hydrolysis

KW - ATPase

KW - P-type ATPase

KW - Radioactive assay

U2 - 10.21769/BioProtoc.4676

DO - 10.21769/BioProtoc.4676

M3 - Journal article

C2 - 37251095

AN - SCOPUS:85162120073

VL - 13

JO - Bio-protocol

JF - Bio-protocol

SN - 2331-8325

IS - 10

M1 - e4676

ER -

ID: 378186096