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Abcam's PicoProbe Acetyl CoA Assay Kit is a highly sensitive assay for determining Acetyl CoA level in a variety of biological samples. In the assay, free CoA is quenched then Acetyl CoA is converted to CoA. The CoA is reacted to form NADH which interacts with PicoProbe to generate fluorescence (Ex=535/Em=587 nm). The assay can detect 10 to 1000 pmol of Acetyl CoA (with detection limit ~0.4 µM) in a variety of samples.
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Acetyl CoA is a central molecule of metabolism. It carries acetate, used in the build-up and breakdown of larger molecules. Acetyl CoA is key in synthetic pathways leading to sesquiterpenes, precursors to cholesterol and other sterols, flavenoids and other polyketides, polyenes and long-chain fatty acids. It is the source of the acetyl group used in histone acetylation. The acetyl group is also incorporated into a variety of other molecules such as acetylcholine, melatonin, heme and TCA cycle intermediates.
|Acetyl CoA Assay Buffer||WM||1 x 25ml|
|Acetyl CoA Enzyme Mix||Purple||1 x 500µl|
|Acetyl CoA Standard (1 µmol) (Lyophilised)||Yellow||1 vial|
|Acetyl CoA Substrate Mix (Lyophilised)||Red||1 vial|
|CoA Quencher||Orange||1 x 1ml|
|Conversion Enzyme||Green||1 x 100µl|
|PicoProbe™ (in DMSO)||Blue||1 x 200µl|
|Quench Remover (Lyophilised)||Clear||1 vial|
Induction of acetyl-CoA in mitochondria by palmitate. Mitochondria were isolated from cells after palmitate treatment (300 μM) at 1, 4 and 16 h, and then used in the concentration assay of acetyl-CoA.
The acetyl-CoA content was determined immediately after isolation of the mitochondria using ab87546. Briefly, acetyl-CoA standard curve was made in the range of 0–100 pM and the correlation coefficient was 0.990 or higher. Protein was removed in the sample using the perchloric acid protocol and the supernatant was neutralized with 3 M KHCO3. The CoASH Quencher and Quencher remover were added into the sample to correct the background generated by free CoASH and succ-CoA. The sample was then diluted with the reaction mix, and the fluorescence signal was measured at Ex/Em = 535/589 nm with Spectra max Gemini XPS (Molecular Devices, Sunnyvale, CA). The relative acetyl-CoA concentration was normalized with the mitochondrial protein.
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