Diacylglycerol Assay Kit (ab242293)
Key features and details
- Detection method: Fluorescent
- Sample type: Cell Lysate
Overview
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Product name
Diacylglycerol Assay Kit -
Detection method
Fluorescent -
Sample type
Cell Lysate -
Product overview
ab242293 measures diacylglycerol (DAG) content in samples by a coupled enzymatic reaction system. First, kinase is used to phosphorylate DAG samples, yielding phosphatidic acid. Next, a lipase is used to hydrolyze phosphatidic acid to glycerol-3-phosphate. Finally, the glycerol-3- phosphate product is oxidized by glycerol-3-phosphate oxidase (GPO), producing hydrogen peroxide which reacts with the kit’s Fluorometric Probe (Ex. 530-560 nm/Em. 585-595 nm).
ab242293 is a simple, fluorometric assay that quantitatively measures total DAG in cell lysates using a 96-well microtiter plate format. Each kit provides sufficient reagents to perform up to 100 assays, including blanks, standards and unknown samples. The kit contains a DAG Standard and has a detection sensitivity limit of ~15 µM.
Properties
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Storage instructions
Store at -80°C. Please refer to protocols. -
Components 100 tests 10X Assay Buffer 1 x 1.5ml DAG Standard 1 x 1ml Enzyme Mixture 3 x 1.75ml Fluorometric Probe 1 x 110µl Kinase Mixture 2 x 1ml Lipase Solution 3 x 1.4ml -
Relevance
Diacylglycerols (DAG) are key intermediates in the biosynthesis of many cellular lipids and play a fundamental role in biochemical signaling. Structurally, diacylglycerol is a glyceride consisting of 2 fatty acid chains linked by a central glycerol backbone. Diacylglycerol acts as a precursor to many lipids (e.g. triglycerides, phospholipids); however, DAG also functions as a second messenger signalling lipid, produced through hydrolysis of PIP2 by phospholipase C, which initiates intracellular Ca2+ release and PKC activation. Ultimately, the PKC enzymes are involved with many modifications to normal cellular physiology, affecting hundreds of substrates.
Images
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DAG Assay Standard Curve.
DAG standard curve was performed according to the Assay Protocol. Background has been subtracted.
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DAG Detection of Lipid Extracts.HEK293 and COS-7 lipid extracts were prepared according to the extraction procedure above. DAG samples were tested according to the Assay Protocol (phosphatidic acid background was determined +/- Kinase Mixture). Negative control values (without DAG) have been subtracted.
