Fumarase Activity Assay Kit (WST-8)
Based on 1 Customer Validation
MCE Fumarase Activity Assay Kit (WST-8) can be used to measure the amount of fumarase present in biological samples.
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Storage :
-20℃, 1 year
Keep away from light and avoid repeated freeze-thaw cycles
NADH should be appropriately aliquoted and stored at -80°C after preparation into solution.
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Description & Advantages
Fumarase (Fumarate Hydratase, FH, EC 4.2.1.2) is an enzyme that catalyzes the reversible reaction of hydration and dehydration of fumarate to malate. It has two forms: mitochondrial and cytosolic forms. The mitochondrial form of fumarase is one of the key enzymes in citric acid cycle or tricarboxylic acid cycle (TCA), while cytosolic form is important for metabolism of amino acids and fumarate. In humans, fumarase deficiency leads to serious health problems such as fetal brain abnormality, hypotonia and renal cell carcinoma. Therefore, accurate measurement of fumarase activity is important for preventing, diagnosis and mechanistic study of fumarase deficiency.
MCE Fumarase Activity Assay Kit (WST-8) can be used to measure the amount of fumarase present in biological samples. The detection principle is as follows: Fumarase catalyzes the conversion of Fumarate into malate. The generated malate is then oxidized to oxaloacetic acid (OAA) in the presence of malate dehydrogenase (MDH). Simultaneously, NAD+ is reduced to NADH. The NADH interacts with the electron-coupled reagent (1-mPMS, 1-Methoxy-5-methylphenazinium Methyl Sulfate), transforms WST-8 into an orange-colored formazan displaying maximum absorption at 450 nm. In conclusion, there is a linear relationship between the amount of formazan generated in the system and the activity of fumarase in the sample.
Features of MCE Fumarase Activity Assay Kit (WST-8)
1. High Sensitivity: At 37˚C for 30 min, with the detection range of 0.7-30 U/L.
2. Wide Linear Range: At 37˚C for 30 min, the assay demonstrates a strong linear correlation within the concentration range of 20-1000 μM.
3. Fast Detection Speed and Broad Applicability: Suitable for various samples such as plasma, serum, red blood cells, tissues, and culture media.
Protocol
Reagent Preparation
1. NADH solution: add 7 mL Detection Buffer to NADH and dissolve completely. Aliquot according to single-use volume and store at -80°C protected from light. Avoid repeated freeze-thaw cycles.
2. Positive Control working solution: dilute Positive Control (100×) 1:100 with Lysis Buffer immediately before use.
3. Reaction working solution: for each well, mix 174 μL Detection Buffer, 2 μL Enzyme Solution, 2 μL Chromogenic Solution, and 2 μL Substrate. Immediately before use, add 20 μL NADH solution per well and mix thoroughly.
Sample Preparation
1. Plasma, serum, or culture supernatant: clear samples may be assayed directly or diluted with Lysis Buffer.
2. Cells: collect approximately 1 × 106 cells, wash with PBS, add 100-200 μL pre-cooled Lysis Buffer, lyse thoroughly on ice, centrifuge at 12,000 × g for 10 min at 4°C, and collect the supernatant.
3. Tissue: weigh 10-30 mg tissue, add 100 μL pre-cooled Lysis Buffer per 10 mg tissue, homogenize on ice, centrifuge at 12,000 × g for 10 min at 4°C, and collect the supernatant.
Assay Procedure
| Well | Sample/Control | Reaction Working Solution |
|---|---|---|
| Blank | 20 μL Lysis Buffer | 180 μL |
| Positive control | 20 μL diluted Positive Control | 180 μL |
| Sample | 20 μL sample | 180 μL |
1. Add blank, positive control, and samples to a clear 96-well plate according to the table.
2. Add 180 μL reaction working solution to each well and mix gently.
3. Immediately measure the initial absorbance A0 at 450 nm.
4. Incubate at 37°C for 30 min protected from light and measure A30 at 450 nm.
Calculation
Calculate ΔA = A30 - A0 and subtract the blank ΔA. Samples should fall within the linear range of the kit; dilute with Lysis Buffer and repeat the assay if necessary. Use the Positive Control to confirm reaction performance and calculate fumarase activity according to the conversion relationship specified in the manual, sample volume, and dilution factor.
Storage
-20℃, 1 year
Keep away from light and avoid repeated freeze-thaw cycles
NADH should be appropriately aliquoted and stored at -80°C after preparation into solution.
Components
| Components | HY-K0321-100 T |
|---|---|
| Enzyme Solution | 200 μL |
| Positive Control (100×) | 20 μL |
| Chromogenic Solution | 200 μL |
| Substrate | 200 μL |
| NADH | 5 mg |
| Lysis Buffer | 20 mL |
| Detection Buffer | 20 mL |
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