Description
Beta-N-Acetylglucosaminidase Activity Assay Kit (BA0053) (BA0053)
The Beta-N-Acetylglucosaminidase Activity Assay Kit (SKU: BA0053) provides a fast, sensitive and non-radioactive colorimetric method for determining beta-N-acetylglucosaminidase (NAG) activity in biological samples. NAG is a lysosomal enzyme involved in the degradation of glycoproteins and glycolipids, cell proliferation and signal transduction. Because of its high molecular weight it cannot be filtered through the glomerular membrane, so elevated urinary NAG activity is an early indication of renal damage due to diabetes mellitus, inflammation, nephrotic syndrome and urinary tract infection, while raised serum levels are associated with various cancers. The assay is based on the cleavage of p-nitrophenol from a synthetic substrate; p-nitrophenol becomes intensely coloured after addition of the stop reagent, and the increase in absorbance at 405nm is directly proportional to the enzyme activity. The homogeneous mix-incubate-measure format can be readily automated for high-throughput screening.
| Product Name: | Beta-N-Acetylglucosaminidase Activity Assay Kit (BA0053) |
| SKU: | BA0053 |
| Detection Method: | Colorimetric |
| Detection Range: | 0.2 to 50 U/L for a 30-minute reaction at 37°C (20 µL sample) |
| Sample Type: | Urine, serum, plasma, cell lysate |
| Species Reactivity: | All |
| Assay Time: | 30 minutes at 37°C |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | The kit is shipped at room temperature. Store the substrate at -20°C and all other components at 4°C upon receipt. |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
This non-radioactive, colorimetric assay is based on the cleavage of p-nitrophenol from a synthetic substrate. p-Nitrophenol becomes intensely coloured after addition of the stop reagent, and the increase in absorbance at 405nm is directly proportional to the enzyme activity.
- Fast and sensitive: linear detection range (20 µL sample) 0.2 to 50 U/L for a 30-minute reaction at 37°C
- High-throughput: homogeneous mix-incubate-measure assay, readily automated on HTS liquid handling systems for thousands of samples per day
- Beta-N-acetylglucosaminidase activity determination in biological samples (e.g. urine, serum, plasma, 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 | Standard preparation: mix 15 µL of the 12.5 mM Nitrophenol standard with 235 µL dH2O to make 750 µM Premix, then dilute as shown in the table. |
| 2 | Transfer 20 µL of each sample into two separate wells (ODSAMPLE and ODSAMPLE BLANK) and 20 µL of each standard into separate wells of a clear flat-bottom 96-well plate. |
| 3 | Add 100 µL of Stop Reagent to the Sample Blank wells. |
| 4 | Add 80 µL of Substrate solution to all standard, sample and sample blank wells; tap plate briefly to mix. |
| 5 | Incubate at 37°C (or desired temperature) for 30 minutes, then add 100 µL Stop Reagent to each standard and sample well (do not add anything more to the sample blank wells). Tap plate briefly to mix and read OD405nm. |
Subtract blank OD (water, #4) from the standard OD values and plot ∆OD against standard concentrations. NAG activity = [(ODSAMPLE − ODSAMPLE BLANK) / (t × Slope)] × n (U/L), where Slope is from the standard linear regression, t is the reaction time (30 minutes) and n is the dilution factor. Unit definition: 1 Unit (U) catalyses the conversion of 1 µmole of p-nitrophenyl N-acetyl-β-D-glucosaminide to p-nitrophenol and N-acetyl-D-glucosamine per minute at 37°C at pH 4.5.
| Component | Quantity | Storage |
| Substrate | 10 mL | -20°C |
| Stop Reagent | 12 mL | 4°C |
| Standard (12.5 mM Nitrophenol) | 1 mL | 4°C |
