Description
Aldehyde Dehydrogenase Assay Kit (BA0234) (BA0234)
Aldehyde dehydrogenases (ALDHs) are a superfamily of oxidoreductases which catalyse the conversion of aldehydes to carboxylic acids. ALDH is crucial in the metabolism of alcohol: alcohol dehydrogenase breaks down ethanol to acetaldehyde, which is toxic to the body and is in turn broken down by ALDH to acetic acid. Imbalances of ALDHs have been linked to both alcoholism and alcohol sensitivity. The Aldehyde Dehydrogenase Assay Kit (SKU: BA0234) is based on the enzymatic conversion of acetaldehyde to acetic acid and NADH by ALDH. The formed NADH is converted into a coloured product, the absorbance of which, measured at 565 nm, is proportional to the enzyme activity in the reaction. The single working-reagent format can be completed in under an hour and is amenable to high-throughput screening.
| Product Name: | Aldehyde Dehydrogenase Assay Kit (BA0234) |
| SKU: | BA0234 |
| Detection Method: | Colorimetric determination at OD565 nm |
| Detection Range: | 0.2 - 25 U/L ALDH in a 96-well plate assay |
| Sample Type: | Biological samples (e.g. serum, cell lysate) |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | The kit is shipped at room temperature. Store all components at -20°C upon receipt. |
| Shelf Life: | 6 months after receipt. |
| Shipping: | Gel Pack |
A quantitative colorimetric assay for aldehyde dehydrogenase activity based on the enzymatic conversion of acetaldehyde to acetic acid and NADH by ALDH. The formed NADH is converted into a coloured product measured at 565 nm, proportional to enzyme activity, with a linear detection range of 0.2 - 25 U/L ALDH.
- Sensitive and accurate. Linear detection range 0.2 - 25 U/L ALDH in a 96-well plate assay.
- Convenient. The assay involves addition of a single working reagent and can be completed in under an hour.
- Robust and amenable to HTS. Homogeneous “mix-incubate-measure” type assay. No wash and reagent transfer steps are involved. Can be readily automated on HTS liquid handling systems for processing thousands of samples per day.
- For quantitative determination of aldehyde dehydrogenase activity in biological samples (e.g. serum, cell lysate, etc).
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 | Equilibrate all components to room temperature and briefly centrifuge tubes before opening. The Working Reagent (WR) should be prepared fresh for each assay run. |
| 2 | Prepare the enzyme in an enzyme buffer, e.g. 100 mM Tris-HCl, pH 8, 100 mM KCl, 0.09% BSA, when required. |
| 3 | Sample preparation: serum samples should be diluted 3-fold or higher prior to the assay; cell lysate can be assayed directly. |
| 4 | Transfer 100 µL H2O (OD_H2O) and 100 µL Calibrator (OD_CAL) solution into wells of a clear flat-bottom 96-well plate. |
| 5 | Transfer 50 µL of each sample to separate wells of the plate. |
| 6 | Prepare enough WR for all wells by mixing, per well, 45 µL Assay Buffer, 8 µL NAD/MTT, 1 µL Diaphorase and 0.25 µL 4x Substrate. |
| 7 | Add 50 µL WR to all Sample wells. Briefly tap plate to mix and read optical density (OD565) at 0 min and at 30 min. |
ALDH Activity = [(OD30 – OD0) / (ε_mtt × l)] × [Reaction Vol / (t × Enzyme Vol)] × n = 3.64 × [(OD30 – OD0) / (ODCal – ODH2O)] × [Reaction Vol / (t × Enzyme Vol)] × n (U/L), where OD30 and OD0 are the OD565 values at 30 and 0 min, ε_mtt is the molar absorption coefficient of reduced MTT, l is the light pathlength from the calibrator, Reaction Vol is 100 µL, Enzyme Vol is 50 µL, t is 30 min and n is the sample dilution factor. Unit definition: 1 U of ALDH catalyses the conversion of 1 µmole of Substrate per min at room temperature and pH 8. If sample activity exceeds 25 U/L, dilute in enzyme buffer and repeat.
| Component | Quantity | Storage |
| Assay Buffer | 12 mL | -20°C |
| Diaphorase | 120 µL | -20°C |
| NAD/MTT | 1 mL | -20°C |
| 4x Substrate (400 mM) | 50 µL | -20°C |
| Calibrator | 1.5 mL | -20°C |
