| 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. |
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| Catalog # | A0504-85 |
| Acetate Assay Kit 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. |
| Key Features | Use as little as 10ul samples. Detection range: 0.20 to 20mM acetate for colorimetric assays and 0.13 to 2mM for fluorimetric assays. |
| Applications | 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. |
| Kit Contents | Assay Buffer: 25ml Enzyme A: 600ul Enzyme B: 120ul |
| Dye Reagent: 120ul ATP: 120ul Standard: 1ml 200mM |
| Storage conditions. The kit is shipped on ice. Store all components at -20°C. Shelf life of three months after receipt. |
| Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information. |
| Fluorimetric Procedure | Sample treatment: 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. |
| 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 400ul 2mM Standard by mixing 4ul 200mM standard with 396ul dH2O. Dilute standard in dH2O as follows. |
| No 2mM STD+H2O Vol (ul) Acetate (mM) |
| 1 100ul+0ul 100 2.0 |
| 2 75ul+25ul 100 1.5 |
| 3 50ul+50ul 100 1.0 |
| 4 25ul+75ul 100 0.5 |
| 5 0ul +100ul 100 0 |
| Transfer 10ul standards and 10ul samples into separate wells of a black flat-bottom 96-well plate. |
| 3. Reaction. Prepare Working Reagent, for each reaction well, by mixing 90ul Assay Buffer, 5ul Enzyme A, 1ul Enzyme B, 1ul Dye Reagent and 1ul ATP. Note: the Working Reagent should be prepared freshly and used within 20 min. Transfer 90ul Working Reagent to each well. Mix immediately and read fluorescence intensity lex/lem=530/585nm. Incubate for 30 min at room temperature and read fluorescence intensity again. |
| Colorimetric Procedure | For colorimetric assays, the detection range is 0 to 20mM acetate. Prepare 0, 4, 8, 12, 16 and 20mM acetate standards in dH2O. Perform the assay the same as for Fluorimetric Procedure, but use a clear flat bottom 96-well plate and read OD 570nm (550-585nm). Notes: If the calculated acetate concentration of a sample is higher than 2mM in fluorimetric assay or 20mM in colorimetric assay, dilute sample in water and repeat the assay. Multiply result by the dilution factor n. |
| Calculation | Subtract time zero values from the time 30 min values and plot the DF or DOD against standard concentrations. Determine the acetate concentration of Sample from the standard curve. Conversions: 1mM acetate equals 5.9 mg/dL, 0.0059% or 59 ppm. |
| General Considerations | 1. SH-containing reagents (e.g. b–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. |
| Materials Required, But Not Provided | Pipetting devices, centrifuge tubes, clear flat-bottom 96-well plates, optical density plate reader; black 96-well plates and fluorescence plate reader. |
| Important Note | This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. |
