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- Target
- Acetate
- Application
- Biochemical Assay (BCA)
- Sample Type
- Serum, Plasma, Food, Agricultural Samples, Environmental Samples
- Specificity
- 0.13 mM
- Characteristics
- Use as little as 10 µL samples. Detection range: 0.20 to 20 mM acetate for colorimetric assays and 0.13 to 2 mM for fluorimetric assays.
- Components
- Assay Buffer: 25 mL. Enzyme A: 600 µL. Enzyme B: 120 µL. Dye Reagent: 120 µL. ATP: 120 µL. Standard: 1 mL 200 mM.
- Material not included
- Pipetting devices, centrifuge tubes, clear flat-bottom 96-well plates, optical density plate reader, black 96-well plates and fluorescence plate reader.
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- Application Notes
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Direct Assays: acetate in biological samples such as serum/plasma, in food, agriculture and environmental samples.
Drug Discovery/Pharmacology: effects of drugs on acetate metabolism. - Comment
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1. SH-containing reagents (e.g. beta-mercaptoethanol, dithiothreitol) are known to interfere in this assay and should be avoided in sample preparation.
2. This assay is based on a kinetic reaction. To ensure identical incubation time, addition of Working Reagent to standard and samples should be quick and mixing should be brief but thorough. Use of a multi-channel pipettor is recommended. - Protocol
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1. Equilibrate all components to room temperature. Briefly centrifuge tubes. During experiment, keep Enzymes in a refrigerator or on ice.
2. Standards and samples: prepare 400 µL 2 mM Standard by mixing 4 µL 200 mM standard with 396 µL dH2O. Dilute standard in dH2O. Transfer 10 µL standards and 10 µL samples into separate wells of a black flat-bottom 96-well plate.
3. Reaction. Prepare Working Reagent, for each reaction well, by mixing 90 µL Assay Buffer, 5 µL Enzyme A, 1 µL Enzyme B, 1 µL Dye Reagent and 1 µL ATP. Note: the Working Reagent should be prepared freshly and used within 20 min. Transfer 90 µL Working Reagent to each well. Mix immediately and read fluorescence intensity ex/em = 530/585nm. Incubate for 30 min at room temperature and read fluorescence intensity again. - Sample Preparation
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Serum and plasma samples can be assayed directly. Acetic acid containing samples such as vinegars should be diluted in the Assay Buffer prior to assay. Samples should be clear, and free of precipitate or particles. If present, precipitate or particles should be removed by filtration or centrifugation.
- Calculation of Results
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Subtract time zero values from the time 30 min values and plot the F or OD against standard concentrations. Determine the acetate concentration of Sample from the standard curve.
Conversions: 1 mM acetate equals 5.9 mg/dL, 0.0059% or 59 ppm. - Restrictions
- For Research Use only
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- Storage
- -20 °C
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- Target
- Acetate
- Target Type
- Amino Acid
- Background
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Quantitative determination of acetic acid or acetate by colorimetric (570nm) or fluorimetric (530nm/590nm) methods.
Procedure: 30 min.
Acetate is a common anion and fundamental to all forms of life. When bound to coenzyme A, it is central to the metabolism of carbohydrates and fats. It is acid form, acetic acid, is produced and excreted by acetic acid bacteria, such as Acetobacter genus and Clostridium acetobutylicum, which are found universally in foodstuffs, water, and soil. Acetic acid is also a component of the vaginal lubrication of humans and other primates, where it appears to serve as a mild antibacterial agent. Acetic acid is the main component of vinegar, and extensively used in food, dyes, paints, glue and synthetic fibres. This assay uses enzyme-coupled reactions to form a colored, fluorescent product. The color absorbance at 570nm or fluorescence intensity at 530nm/585nm is directly proportional to the acetate concentration in the sample.
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