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Coenzyme A (CoA) is an essential metabolic cofactor synthesized from cysteine, pantothenate, and ATP. It is involved in many important biological activities including the synthesis and oxidation of fatty acids, pyruvate oxidation in the citric acid cycle and many others. One of CoA’s most crucial roles is the carrying and transferring of acyl groups. CoA levels can be altered during starvation, and in conditions such as cancer, diabetes, and alcoholism.

Coenzyme A Assay Kit provides a simple, reproducible, and sensitive tool of measuring Coenzyme A concentration in cell lysates, plasma, serum and other biological liquid samples. This assay employs a coupled enzymatic reaction system that combines multiple steps. Initially, CoA is converted to acyl-CoA which is oxidized to enoyl-CoA and hydrogen peroxide (H2O2). Peroxidase then catalyzes the reaction of hydrogen peroxide with the Colorimetric Probe to form a pink colored product. The optical density measured at 570nm is directly proportional to the concentration of Coenzyme A present in the sample.

• Microplate reader capable of measuring absorbance at 570 nm
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Tubes to prepare sample dilutions
• Incubator at 37°C
• 15 ml and 50 ml conical tubes
• MilliQ water or another type of double distilled water (ddH2O)

1. Add 40 µL of the diluted Coenzyme A Standards or samples to the 96-well microtiter plate.
2. Initiate the reaction by adding 80 µL of Conversion Solution to each well.
3. Mix well. Incubate for 30 minutes at 37°C, protect from light.
4. Add 80 µL of Developing Solution to each well. Mix well, incubate for 30 minutes at 37°C, protect from light.
5. Measure the absorbance at 570nm using a plate reader.