Description
Aldehyde Dehydrogenase Inhibitor Screening Kit (BA0249) (BA0249)
The Aldehyde Dehydrogenase Inhibitor Screening Kit (SKU: BA0249) provides a quantitative colorimetric method for determining the inhibitory activity of test compounds against aldehyde dehydrogenase (ALDH) at 565 nm. Aldehyde dehydrogenases are a superfamily of oxidoreductases that catalyse the conversion of aldehydes to carboxylic acids and are crucial in alcohol metabolism, converting toxic acetaldehyde to acetic acid; imbalances have been linked to alcoholism and alcohol sensitivity, while heightened ALDH activity in cancer stem cell populations makes ALDH inhibition a promising anti-cancer approach. The assay is based on the enzymatic conversion of acetaldehyde to acetic acid and NADH by ALDH, whereupon the NADH reduces a formazan reagent into a coloured product whose absorbance at 565 nm is proportional to enzyme activity. Percent inhibition of a test compound is determined by comparing the activity of ALDH treated with a test compound to that of untreated ALDH. The homogeneous mix-incubate-measure assay can be completed in under an hour and is readily automated for high-throughput screening of thousands of samples per day.
| Product Name: | Aldehyde Dehydrogenase Inhibitor Screening Kit (BA0249) |
| SKU: | BA0249 |
| Detection Method: | Colorimetric (OD565nm) |
| Sample Type: | Purified aldehyde dehydrogenase and test compounds |
| Species Reactivity: | All |
| Assay Time: | Under one hour |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20°C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A quantitative colorimetric inhibitor screening assay for aldehyde dehydrogenase (ALDH). ALDH converts acetaldehyde to acetic acid and NADH, which reduces a formazan reagent to a coloured product measured at 565 nm; percent inhibition is derived by comparison with untreated ALDH.
- Rapid and reliable. Can be completed in under an hour.
- Homogeneous and convenient. Mix-incubate-measure type assay. No wash and reagent transfer steps are involved.
- Robust and amenable to HTS: can be readily automated on HTS liquid handling systems for processing thousands of samples per day.
- HTS for inhibitor screening and evaluation of ALDH inhibitors
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 assay is based on an enzyme-catalysed kinetic reaction. To ensure identical incubation time, addition of Working Reagent should be quick and mixing brief but thorough; a multi-channel pipettor is recommended. Note: neither the ALDH enzyme nor a control inhibitor is included in the kit. |
| 2 | Reagent preparation: equilibrate all components to room temperature. Keep 4Substrate and Diaphorase on ice. Pre-warm Assay Buffer to 25°C. Prepare the Reaction Mix fresh and use within two hours. |
| 3 | Sample preparation (optimised for ALDH from baker's yeast): for other species, determine the Km and adjust the substrate volume so the final concentration in the 100 µL reaction is near the Km. Dilute purified ALDH to 22 U/mL using assay buffer. Dissolve test compounds in a solvent of choice (DMSO at 5 v/v% or less in the final reaction will not interfere). |
| 4 | Transfer 45 µL of 22 U/mL ALDH into separate wells. Reserve two ALDH wells for the Blank (no substrate) and Control (no inhibitor). |
| 5 | To the Control and Blank wells add 5 µL of the solvent used for the test compounds. To the remaining ALDH wells add 5 µL of the test compounds. |
| 6 | Prepare enough 1 Substrate by diluting 4 Substrate 4-fold in dH2O (each well needs 1 µL). Prepare sufficient Reaction Mix (RM) by mixing, per well (except blank): 45 µL Assay Buffer, 8 µL NAD/MTT, 1 µL Diaphorase and 1 µL 1 Substrate. Prepare Blank Reaction Mix (BRM) per blank well: 45 µL Assay Buffer, 8 µL NAD/MTT and 1 µL Diaphorase (no 1 Substrate). Add 50 µL BRM to the Blank well and 50 µL RM to the remaining wells. Tap to mix briefly and thoroughly. |
| 7 | Incubate the plate for 30 minutes at room temperature and read optical density at 565 nm. |
ALDH inhibition for a test compound: % Inhibition = (1 – ∆ODTestCpd / ∆ODNoInhibitor) × 100%, where ∆ODTestCpd is the OD565nm value of a test compound minus the OD565nm value of the Blank well at 30 min, and ∆ODNoInhibitor is the OD565nm value of the Control minus the OD565nm value of the Blank well at 30 min.
| Component | Quantity | Storage |
| Assay Buffer | 12 mL | -20°C |
| NAD/MTT | 1 mL | -20°C |
| Diaphorase | 120 µL | -20°C |
| 4Substrate (400 mM) | 50 µL | -20°C |
