Description
Invertase Activity Assay Kit (BA0123) (BA0123)
This Invertase Assay Kit (SKU: BA0123) offers a convenient and ultra-sensitive colorimetric and fluorimetric means of measuring invertase and sucrase activity. Invertase (β-fructofuranosidase, EC 3.2.1.26) catalyses the hydrolysis of sucrose to fructose and glucose, and a wide range of microorganisms produce invertase in order to utilise sucrose as a nutrient. The assay finds wide application across environmental (e.g. soil), agricultural and food industries. In the reaction, invertase cleaves sucrose to form fructose and glucose, and the glucose produced is then measured by a colorimetric (570 nm) or fluorimetric (λex/em = 530/585 nm) method. The assay is simple, sensitive, stable and readily adaptable to high-throughput formats.
| Product Name: | Invertase Activity Assay Kit (BA0123) |
| SKU: | BA0123 |
| Detection Method: | Colorimetric (570 nm) or fluorimetric (λex/em = 530/585 nm) |
| Detection Range: | As low as 0.007 U/L invertase activity |
| Sample Type: | Biological and environmental samples (e.g. soil) |
| Species Reactivity: | All |
| Assay Time: | Approximately 40 minutes (20 min enzyme reaction plus 20 min glucose determination) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all components at -20°C. |
| Shelf Life: | 6 months after receipt. |
| Shipping: | Gel Pack |
Invertase cleaves sucrose to form fructose and glucose; the glucose produced is measured colorimetrically at 570 nm or fluorimetrically at λex/em = 530/585 nm. One unit of invertase catalyses the formation of 1 µmole of glucose per minute at pH 4.5 under the assay conditions.
- Safe, non-radioactive assay.
- Sensitive and accurate, quantifying invertase activity as low as 0.007 U/L.
- Homogeneous and convenient 'mix-incubate-measure' type assay with no wash or reagent transfer steps.
- Robust and amenable to high-throughput screening, readily automated for thousands of samples per day.
- Invertase and sucrase activity determination in biological and environmental (e.g. soil) samples.
- Evaluation and screening for invertase 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 | Interference: thiols (β-mercaptoethanol, dithioerythritol, etc.) above 10 µM interfere with this assay and should be avoided; glucose present in the sample should be removed by dialysis or membrane filtration. |
| 2 | Bring all components to room temperature and briefly centrifuge tubes before opening; dilute the 10x Reaction Buffer and 10x Sucrose ten-fold by mixing 1 volume of reagent with 9 volumes of distilled water and use the diluted reagents for all assays. |
| 3 | For the glucose standard curve, mix 5 µL Glucose Standard with 828 µL distilled water (final 100 µM), dilute as described in the standard table and transfer 40 µL of each standard to separate wells of a clear flat-bottom 96-well plate. |
| 4 | Transfer 40 µL of each sample to separate wells and use 40 µL of diluted Reaction Buffer as a sample control. |
| 5 | Enzyme reaction: add 5 µL of the diluted Sucrose to each well, tap to mix and incubate for 20 minutes at the desired temperature (e.g. 30°C). |
| 6 | Glucose determination: prepare Working Reagent in bulk by mixing, per well, 95 µL Assay Buffer, 1 µL Enzyme Mix and 1 µL Dye Reagent, then add 90 µL Working Reagent to each well and immediately tap to mix. |
| 7 | Incubate for 20 minutes in the dark and read the optical density at 570 nm. |
| 8 | For fluorimetric assays the procedure is the same except that a black flat-bottom 96-well plate is used, glucose standards should be at 20, 12, 6 and 0 µM, and fluorescence intensity is measured at λex/em = 530/585 nm. |
| 9 | For soil samples, weigh about 100 mg of soil into a 1.5-mL tube, add 880 µL diluted Reaction Buffer and 120 µL diluted sucrose, homogenise, remove a 200 µL 'time zero' aliquot as a control, incubate the reaction for 1 hour at 30 or 37°C, then centrifuge and assay the supernatant for glucose. |
Plot the glucose standard curve and determine its slope (µM^-1). Invertase Activity (U/L) = (R_SAMPLE - R_CONTROL) / (Slope x t), where R_SAMPLE and R_CONTROL are the OD or fluorescence values of the sample and sample control (Reaction Buffer) and t is the incubation time (20 min). One unit of invertase catalyses the formation of 1 µmole glucose per minute at pH 4.5. If the signal exceeds the value for the 100 µM glucose (colorimetric) or 20 µM (fluorimetric) standard, dilute the sample in 1-fold Reaction Buffer and repeat, multiplying the result by the dilution factor.
| Component | Quantity | Storage |
| 10x Reaction Buffer (pH 4.5) | 12 mL | -20°C |
| 10x Sucrose | 1.5 mL | -20°C |
| Assay Buffer | 10 mL | -20°C |
| Enzyme Mix | 120 µL | -20°C |
| Glucose Standard | 1 mL | -20°C |
| Dye Reagent | 120 µL | -20°C |
