Description
Coenzyme A (CoA) Assay Kit (Colorimetric/Fluorometric) (BA0093) (BA0093)
The Coenzyme A (CoA) Assay Kit (Colorimetric/Fluorometric) (SKU: BA0093) provides a simple, two-step and high-throughput procedure for measuring coenzyme A. Coenzyme A is involved in many biological activities, including the synthesis and oxidation of fatty acids and pyruvate oxidation in the citric acid cycle, and one of its most crucial roles is the carrying and transferring of acyl groups. In this assay, the first step enzymatically converts CoA to acyl-CoA and the second step oxidises the acyl-CoA, producing enoyl-CoA and hydrogen peroxide. The resulting hydrogen peroxide reacts with a specific dye to form a pink coloured product; the optical density at 570 nm, or fluorescence intensity at 530/585 nm, is directly proportional to the CoA concentration in the sample.
| Product Name: | Coenzyme A (CoA) Assay Kit (Colorimetric/Fluorometric) (BA0093) |
| SKU: | BA0093 |
| Detection Method: | Colorimetric or Fluorometric |
| Detection Range: | Colorimetric 5 to 1000 uM; fluorometric 3 to 100 uM CoA |
| Sample Type: | Serum, plasma, milk, solid samples and other biological samples |
| Species Reactivity: | All |
| Assay Time: | Two 30 minute incubations at room temperature |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
Quantitative colorimetric or fluorometric determination of coenzyme A. CoA is converted to acyl-CoA and then oxidised to produce hydrogen peroxide, which reacts with a dye to form a pink product read at 570 nm or by fluorescence at 530/585 nm.
- Sensitive; uses 10 uL sample with a colorimetric range 5 to 1000 uM and fluorometric range 3 to 100 uM CoA
- Convenient room-temperature mix-and-read procedure
- Readily automated for high-throughput analysis
- Two-step enzymatic detection
- Assays of coenzyme A in a variety of biological samples
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Reagent preparation. Reconstitute Enzyme A by adding 120 uL distilled water to the Enzyme A tube, ensuring it is fully dissolved by pipetting up and down, and incubate at room temperature for 15 minutes; store reconstituted Enzyme A at -20C and use within 2 months. |
| 2 | Sample preparation (colorimetric). Assay serum and plasma directly. Homogenise milk and solid samples in 5% isopropanol and 5% Triton X-100 in water, followed by filtration through a 0.45 um PTFE syringe filter. Avoid SH-containing reagents above 5 uM, sodium azide, EDTA and SDS. |
| 3 | Equilibrate all components to room temperature and briefly centrifuge tubes; keep thawed tubes on ice during the assay. The thawed standard should be clear and colourless; if the substrate is turbid, bring it to 37C and gently swirl until clear. |
| 4 | Standards. Prepare a 1000 uM stock by diluting 5 uL of the 100 mM standard with 495 uL Assay Buffer, then dilute in Assay Buffer as shown in the dilution table. Transfer 10 uL diluted standards into separate wells of a clear flat-bottom 96-well plate, and 10 uL of each sample into separate wells. |
| 5 | ACS reaction. Prepare working reagent by mixing, per well, 40 uL Assay Buffer, 1 uL Enzyme A, 5 uL Substrate and 1 uL ATP. Add 40 uL to each well, tap to mix and incubate at room temperature for 30 minutes. |
| 6 | ACOD reaction. Prepare working reagent by mixing, per well, 55 uL Assay Buffer, 1 uL Enzyme B and 1 uL Dye Reagent. Add 50 uL to each well, tap to mix and incubate 30 minutes at room temperature protected from light. Read optical density at 570 nm (550-585 nm). |
| 7 | Fluorometric assay. The procedure is similar, using 0, 30, 60 and 100 uM standards in a black 96-well plate; read fluorescence at excitation 530 nm and emission 585 nm. |
Subtract the blank value (standard #4) from the standard values and plot dOD or dF against standard concentrations to determine the slope. [CoA] = (R-sample - R-blank) / slope x n (uM), where R is the optical density or fluorescence reading and n is the sample dilution factor. If the calculated concentration exceeds 1000 uM (colorimetric) or 100 uM (fluorometric), dilute the sample in Assay Buffer, repeat and multiply by the dilution factor.
| Component | Quantity | Storage |
| Assay Buffer | 20 mL | -20C |
| Dye Reagent | 120 uL | -20C |
| Enzyme A | Dried | -20C |
| Enzyme B | 120 uL | -20C |
| Substrate | 600 uL | -20C |
| Standard | 50 uL | -20C |
| ATP | 120 uL | -20C |
