Radiolabeled Atp
Radiolabeled Atp - Related Videos
08:05
Assaying Protein Kinase Activity with Radiolabeled ATP
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Cited by 15 •
2017
Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([γ-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.
01:31
ATP Yield
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2019
Cellular respiration produces 30 - 32 ATP per glucose molecule. Although most of the ATP results from oxidative phosphorylation and the electron transport chain (ETC), 4 ATP are gained beforehand (2 from glycolysis and 2 from the citric acid cycle). The ETC is embedded in the inner mitochondrial membrane and is comprised of four main protein complexes and an ATP synthase. NADH and FADH2 pass electrons to these complexes, which pump protons into the intermembrane space. This distribution of...
08:32
The ATP Bioluminescence Assay
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2023
In fireflies, the luciferase enzyme converts a compound called luciferin into oxyluciferin, and produces light or “luminescence” as a result. This reaction requires energy derived from ATP in order to proceed, so researchers have exploited the luciferase-luciferin interaction to gauge ATP levels in cells. Given ATP’s role as the cell’s currency of energy, the ATP bioluminescence assay can provide insight into cellular metabolism and overall cell health.In this video, JoVE discusses cellular...
01:08
Hydrolysis of ATP
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2019
The bonds of adenosine triphosphate (ATP) can be broken through the addition of water, releasing one or two phosphate groups in an exergonic process called hydrolysis. This reaction liberates the energy in the bonds for use in the cell—for instance, to synthesize proteins from amino acids. If one phosphate group is removed, a molecule of ADP—adenosine diphosphate—remains, along with inorganic phosphate. ADP can be further hydrolyzed to AMP—adenosine monophosphate—by the removal of a second...
03:28
Labeling Murine T Helper Lymphocytes using Radiolabeled Monoclonal Antibodies
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2025
In this video, we demonstrate the intracellular radiolabeling of T helper cells using radioactive isotope-conjugated monoclonal antibodies for non-invasive positron emission tomography imaging.
FAQs
Kinase activity is inferred from the appearance of radioactivity in the reaction product. When an enzyme transfers a labeled phosphate from ATP to a substrate, the product retains a detectable signal. Measuring that signal across reactions can reveal reaction rates and help evaluate substrate specificity and enzyme activity.
The isotope is commonly incorporated into an ATP phosphate group, so its location determines what can be tracked. If an enzyme transfers that phosphate to a substrate, labeling follows the phosphorylation event. During nucleotide-dependent synthesis, the labeled ATP can instead help trace incorporation into the newly formed product.
Radiation-sensitive methods make labeled reaction products detectable after ATP participates in a biochemical process. The resulting signal allows researchers to determine whether labeling occurred and compare its extent between reactions. These comparisons support analysis of reaction rates, substrate specificity, phosphorylation, and other aspects of enzyme activity.
A typical workflow supplies radiolabeled ATP to an enzyme reaction in which the nucleotide is transferred to a substrate or used during synthesis. After the reaction, researchers examine the resulting products with a radiation-sensitive detection method. The detected labeling is then related to enzyme activity, reaction rate, or product formation.
During DNA or RNA synthesis, radiolabeled ATP can serve as the labeled nucleotide participating in product formation. Detection of radioactivity in the resulting nucleic-acid product indicates that the nucleotide was used during synthesis. This application enables researchers to follow labeling in nucleic-acid production rather than focusing only on phosphate transfer to a separate substrate.
The technique supports studies of phosphorylation, nucleotide metabolism, and enzyme activity in addition to kinase assays and nucleic-acid labeling. By tracking ATP participation and examining labeled products, researchers can investigate how enzymes use nucleotides, which substrates they act on, and how rapidly the associated biochemical reactions proceed.