Atpase Activity Assay
Atpase Activity Assay - Related Videos
07:38
Measuring In Vitro ATPase Activity for Enzymatic Characterization
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Cited by 27 •
2016
We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.
06:09
Assaying the Kinase Activity of LRRK2 in vitro
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Cited by 3 •
2012
Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction...
10:28
A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
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Cited by 22 •
2019
A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 µL per well. The platform should be applicable to virtually any ADP producing enzyme.
03:20
XTT Assay: A Colorimetric Assay to Assess Cell Viability and Mitochondrial Activity
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2023
This article describes the protocol for XTT assay which is a colorimetric assay to assess cell viability and mitochondrial activity.
12:48
Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays
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Cited by 7 •
2013
We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.
FAQs
The hydrolysis rate reflects how rapidly the tested enzyme or motor protein uses ATP as an energy source. Measuring this rate provides a functional readout that can help characterize protein behavior and cellular energy use. Comparing rates under different assay conditions or between protein variants reveals changes in ATP-driven activity without relying only on protein presence or abundance.
ATPase activity can be quantified by tracking inorganic phosphate produced during ATP hydrolysis, measuring the decline in ATP, or monitoring a coupled absorbance or fluorescence change. These approaches measure different signals associated with the same reaction. The selected readout determines how activity is followed and enables researchers to express enzyme performance through a measurable change over time.
Cofactors and inhibitors help reveal which conditions support or restrict ATP hydrolysis, while mutant proteins allow direct comparison with an altered version of the protein. Changes in the measured activity can therefore provide evidence about functional requirements, regulatory effects, or consequences of sequence changes. This makes the assay useful for connecting biochemical activity with protein function.
A typical workflow begins by selecting the enzyme or motor protein and supplying ATP under the conditions being examined. The reaction is then followed through phosphate production, ATP depletion, or a coupled absorbance or fluorescence signal. Researchers use the resulting measurement to determine hydrolysis activity and compare samples, conditions, cofactors, inhibitors, or protein variants.
Researchers use this assay when they need to evaluate how a protein converts ATP hydrolysis into measurable activity. It supports studies of membrane transport, molecular motors, signaling proteins, and other ATP-driven biological processes. The method is especially informative when the goal is to compare protein variants or test how cofactors and inhibitors influence function.
ATPase measurements connect biochemical energy use with larger cellular processes. In membrane transport, they can help investigate proteins associated with energy-dependent movement; in molecular motors, they support analysis of ATP-driven function; and in signaling studies, they help evaluate ATP-utilizing proteins. These results provide a functional perspective on how proteins contribute to biological activity.