Description
alpha-L- Fucosidase Assay Kit (AFU) (BA0023) (BA0023)
The alpha-L-Fucosidase Assay Kit (AFU) (SKU: BA0023) is a non-radioactive, colorimetric assay for the quantitative determination of alpha-L-fucosidase activity in biological samples. alpha-L-Fucosidase catalyses the breakdown of L-fucose, and its serum activity is of considerable clinical interest: a genetic deficiency causes the storage disease fucosidosis, low activity has been linked to ovarian carcinoma, and elevated activity is observed in diabetes, hepatitis, cirrhosis and several carcinomas. The assay is based on the cleavage of 4-nitrophenol from a synthetic substrate, with the nitrophenol becoming intensely coloured after the addition of a stop reagent. The increase in absorbance at 405 nm is directly proportional to the enzyme activity. The simple add-mix-read procedure allows reliable quantitation within twenty minutes and is fully compatible with high-throughput liquid handling.
| Product Name: | alpha-L- Fucosidase Assay Kit (AFU) (BA0023) |
| SKU: | BA0023 |
| Detection Method: | Colorimetric kinetic; cleavage of 4-nitrophenol from a synthetic substrate (OD405nm) |
| Detection Range: | 1 to 100 U/L for a 20-minute reaction (10 µL sample) |
| Sample Type: | Plasma, serum, tissue, cell lysate |
| Species Reactivity: | All |
| Assay Time: | 20 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all components at -20°C upon receipt. |
| Shelf Life: | 12 months after receipt. |
| Shipping: | Room Temperature |
A non-radioactive, colorimetric kinetic assay for the quantitative determination of alpha-L-fucosidase (AFU) activity. The assay is based on the cleavage of 4-nitrophenol from a synthetic substrate; the increase in absorbance at 405 nm after the addition of the stop reagent is directly proportional to enzyme activity. Linear detection range is 1 to 100 U/L for a 20-minute reaction.
- High sensitivity and wide linear range: 1 to 100 U/L for a 20-minute reaction (10 µL sample).
- Homogeneous and simple: an add-mix-read procedure gives reliable quantitation within 20 minutes.
- Robust and amenable to HTS: all reagents are compatible with high-throughput liquid handling.
- alpha-L-Fucosidase activity determination in biological samples such as plasma, serum, tissue and cell lysate.
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 | Sample preparation: assay serum and plasma directly. Homogenise tissue (50 mg) in ~200 µL 50 mM potassium phosphate (pH 7.5) and centrifuge at 10,000 g for 15 min at 4°C. Prepare cell lysates similarly. Samples may be stored at -80 to -20°C for at least one month. |
| 2 | Standard preparation: mix 10 µL of 12.5 mM nitrophenol standard with 490 µL dH2O to make 250 µM standard, then dilute as shown in the standards table. |
| 3 | Transfer 100 µL of each standard into wells of a clear flat-bottom 96-well plate. |
| 4 | Transfer 20 µL of each sample into separate wells and add 80 µL Substrate to each. Tap briefly to mix. |
| 5 | Incubate at 25°C (or the desired temperature) for 20 minutes, then add 100 µL Stop Reagent to each well and tap to mix. |
| 6 | Read OD405nm. For coloured or opaque samples, run a sample blank (20 µL sample + 80 µL dH2O). |
Subtract the blank OD (water, standard #4) from the standard OD values and plot ΔOD against concentration to determine the slope. AFU Activity = [(ODsample - ODblank) / (Time x Slope)] x (Reaction Vol / Sample Vol) x n = [(ODsample - ODblank) / Slope] x (1/4) x n (U/L). One unit converts 1 µmole of 4-nitrophenyl-alpha-L-fucopyranoside to 4-nitrophenol and alpha-L-fucose per minute at 25°C and pH 5.3.
| Component | Quantity | Storage |
| Substrate Buffer | 10 mL | -20°C |
| Stop Reagent | 12 mL | -20°C |
| Standard (12.5 mM Nitrophenol) | 1 mL | -20°C |
