Description
Fumarase Activity Assay Kit (Colorimetric) (BA0105) (BA0105)
The Fumarase Activity Assay Kit (Colorimetric) (SKU: BA0105) provides a non-radioactive colorimetric method for determining fumarase (fumarate hydratase, EC 4.2.1.2) activity. Fumarase catalyses the reversible hydration of fumarate to malate and facilitates a key energy-producing transition in the citric acid cycle. The assay couples the reaction to the reduction of the tetrazolium salt MTT in an NADH-linked reaction, producing a reduced MTT form that absorbs maximally at 565 nm. The increase in absorbance at 565 nm is proportional to enzyme activity. This homogeneous mix-incubate-measure format is readily automated for high-throughput screening.
| Product Name: | Fumarase Activity Assay Kit (Colorimetric) (BA0105) |
| SKU: | BA0105 |
| Detection Method: | Colorimetric kinetic (565 nm) |
| Detection Range: | 0.4 - 70 U/L (30 min reaction at 37C) |
| Sample Type: | Plasma, serum, erythrocytes, tissue and culture media |
| Species Reactivity: | All |
| Assay Time: | 30 minutes (kinetic) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A colorimetric kinetic assay for fumarase activity suitable for a range of biological samples, run at room temperature or 37C.
- Fast and sensitive with a linear detection range of 0.4 - 70 U/L for a 30-minute reaction at 37C
- Convenient, high-throughput homogeneous mix-incubate-measure format
- Readily automated for high-throughput screening
- Fumarase activity determination in biological samples such as plasma, serum, erythrocytes, tissue and culture media
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 | This is a kinetic assay; add Working Reagent quickly and mix briefly but thoroughly using a multi-channel pipettor. Assays can be run at 25C or 37C (37C recommended). Serum and plasma are assayed directly; homogenise tissue in cold 50 mM potassium phosphate (pH 7.5) and centrifuge. |
| 2 | Equilibrate reagents to the reaction temperature and centrifuge tubes briefly. |
| 3 | Transfer 100 uL water and 100 uL Calibrator into separate wells of a clear flat-bottom 96-well plate. |
| 4 | Transfer 20 uL water into one well (blank) and 20 uL of each sample into separate wells. |
| 5 | Prepare Working Reagent per 96-well assay from 74 uL Assay Buffer, 8 uL NAD/MTT, 5 uL Substrate, 1 uL Enzyme A and 1 uL Enzyme B. Add 80 uL to all sample and blank wells and tap to mix. |
| 6 | Read OD565nm at 10 minutes and at 40 minutes. |
Subtract OD10 from OD40 for each sample (delta-ODS) and for the blank (delta-ODB). Fumarase activity is calculated from the difference (delta-ODS - delta-ODB), the light path length derived from the calibrator, the reaction and sample volumes (100 uL and 20 uL), the reaction time (30 min recommended at 37C) and the dilution factor n. One unit converts 1 umole of fumarate to L-malate per minute at pH 7.8. If activity exceeds 70 U/L dilute and repeat; for activity below 1 U/L extend incubation to 2 hours.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| NAD/MTT | 1 mL | -20C |
| Substrate | 600 uL | -20C |
| Enzyme A | 120 uL | -20C |
| Enzyme B | 120 uL | -20C |
| Calibrator | 1.5 mL | -20C |
