Description
beta-Glucuronidase Assay Kit (BA0217) (BA0217)
The beta-Glucuronidase Assay Kit (SKU: BA0217) provides a highly sensitive fluorimetric method for the quantitative determination of beta-glucuronidase activity in biological and bacterial samples. Beta-glucuronidase catalyses the hydrolysis of beta-glucuronide bonds and plays an important role in drug metabolism, bilirubin clearance and gene-reporter systems where GUSB is used to track gene expression. The assay employs a beta-D-glucuronide substrate that fluoresces upon hydrolysis and is read at 365/450 nm, allowing detection from 1x10-4 U/L to 8 U/L using only 10 microlitres of sample within 30 minutes. It has been validated to work with both animal and E. coli derived beta-glucuronidase and is presented as a homogeneous mix-incubate-measure format. The procedure is readily automated for high-throughput screening of thousands of samples per day and can be miniaturised for 384- or 1536-well plates.
| Product Name: | beta-Glucuronidase Assay Kit (BA0217) |
| SKU: | BA0217 |
| Detection Method: | Fluorimetric, kinetic; read at lambda ex/em = 365/450 nm |
| Detection Range: | 1x10-4 U/L to 8 U/L beta-glucuronidase activity |
| Sample Type: | Tissue lysate, cell lysate, serum, bacterial samples |
| Species Reactivity: | All |
| Assay Time: | Approximately 45 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all kit components at -20 degrees C. |
| Shelf Life: | 6 months after receipt |
| Shipping: | Room Temperature |
Beta-glucuronidase is an enzyme that catalyses the hydrolysis of beta-glucuronide bonds. In humans it hydrolyses beta-D-glucuronic acids from glycoproteins and mucopolysaccharides such as heparan sulfate, and its activity is important for drug metabolism because drugs are often conjugated to glucuronic acid for more water-soluble delivery. Bilirubin clearance is also facilitated by glucuronidation, and the enzyme is frequently used as a reporter to track gene expression. This kit uses a beta-D-glucuronide substrate that fluoresces upon hydrolysis and is read at 365/450 nm, with fluorescence proportional to enzyme activity.
- Highly sensitive: the fluorimetric format detects 1x10-4 U/L to 8 U/L beta-glucuronidase activity in a 96-well plate with only 10 microlitres of sample within 30 minutes.
- Versatile: validated to work with both animal and E. coli derived beta-glucuronidase.
- High-throughput: homogeneous mix-incubate-measure type assay that can be readily automated to assay thousands of samples per day.
- Quantitative determination of beta-glucuronidase enzyme activity in tissue and bacterial samples.
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 a kinetic reaction, so addition of substrate to samples should be quick and mixing brief but thorough; use of a multi-channel pipettor is recommended and assays can be run at room temperature or 37 degrees C. |
| 2 | For sample preparation, avoid surfactants such as SDS or Triton 100-X. |
| 3 | For tissue, rinse in phosphate buffered saline (pH 7.4) to remove blood prior to dissection, then homogenise 50 mg tissue in approximately 200 microlitres of lysis buffer. |
| 4 | For cell lysate, collect cells by centrifugation at 2,000 x g for 5 minutes at 4 degrees C, use a rubber policeman rather than proteolytic enzymes for adherent cells, then homogenise or sonicate in cold lysis buffer; samples may be stored at -20 to -80 degrees C for at least one month. |
| 5 | Equilibrate all kit reagents to room temperature and invert or gently vortex to ensure they are mixed. |
| 6 | Prepare a 100 uM Premix by mixing 5 microlitres of the provided Standard with 995 microlitres of distilled water, then prepare standards as shown in the standard preparation table. |
| 7 | For tissue lysates dilute samples 5-10 fold with deionised water prior to assay, and for serum samples a 15-fold dilution may be required. |
| 8 | Transfer 10 microlitres of standards and 10 microlitres of samples into wells of a black flat-bottom 96-well plate. |
| 9 | Add 40 microlitres of substrate to each well, tap the plate to mix and incubate at room temperature or 37 degrees C for 30 minutes. |
| 10 | Add 15 microlitres of stop reagent to each well, tap the plate to mix and incubate for 15 minutes. |
| 11 | Read fluorescence at lambda ex/em = 365/450 nm. |
| 12 | This assay procedure can be readily miniaturised for 384- or 1536-well plates. |
BG Activity = [(F_SAMPLE - F_BLANK) / (Time x Slope)] x n (U/L), where F_SAMPLE is the RFU value for each sample and F_BLANK is the RFU value of the water (standard #4). Slope is the slope of the standard curve and Time is the incubation time (30 min). n is the sample dilution factor. Unit definition: 1 Unit (U) of beta-glucuronidase will catalyse the conversion of 1 micromole of the fluorescent glucuronide at 37 degrees C and pH 5.0. Note: if a sample exceeds 8 U/L calculated activity, use a shorter incubation time or dilute samples in water and repeat; for samples below 0.2 U/L, the incubation time can be extended up to 2 hours for greater sensitivity.
| Component | Quantity | Storage |
| Substrate | 4 mL | -20 degrees C |
| Standard | 100 uL | -20 degrees C |
| Stop Reagent | 1.5 mL | -20 degrees C |
