Description
Phytase Assay Kit (BA0222) (BA0222)
The Phytase Assay Kit (SKU: BA0222) provides a simple, sensitive method for measuring phytase activity in agricultural and biological samples. Phytase catalyses the hydrolysis of phosphoester bonds on phytic acid (myo-inositol hexakisphosphate), releasing inositol and phosphate; phytic acid is the major phosphate storage reservoir in plants and phytase is abundant in grains such as wheat and barley. The hydrolysis of phytic acid is a crucial aspect of animal nutrition in single-stomached animals, and a lack of phytase can lead to excessive phosphorus leaching into the environment from undigested phytic acid. This assay is based on a Malachite Green phosphate reaction, with the colour intensity measured at 620 nm being proportional to the phosphate released from phytic acid. The homogeneous mix-and-measure format allows quantification of phytase activity within sixty minutes and offers a wide detection range with no complex detection reagents to mix.
| Product Name: | Phytase Assay Kit (BA0222) |
| SKU: | BA0222 |
| Detection Method: | Colorimetric detection at 620 nm (readable 600-660 nm) based on a Malachite Green phosphate assay. The colour intensity is proportional to the amount of phosphate released from phytic acid by phytase. |
| Detection Range: | Detection range 0.01 to 20 U/L phytase in a 96-well plate assay |
| Sample Type: | Agricultural and biological samples containing phytase (e.g. grain and plant extracts) |
| Species Reactivity: | All |
| Assay Time: | Within 60 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all components at 4°C upon receipt. |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
This assay measures phytase activity by quantifying the phosphate released from phytic acid using a Malachite Green colour reaction. Phytase in the sample hydrolyses phytic acid to release phosphate, which is then detected by a Colour Development Reagent to give a colour whose intensity at 620 nm is proportional to the phosphate liberated and hence to phytase activity, quantified against a phosphate standard curve.
- Simple. No complex detection reagents to mix.
- High sensitivity and wide detection range: 0.01 to 20 U/L phytase in a 96-well plate assay.
- Fast and convenient: homogeneous mix-and-measure assay allowing quantification of phytase activity within 60 minutes.
- Direct assays of phytase activity in agricultural and biological 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 | Sample preparation: avoid phosphate-containing detergents and buffers, as the assay is extremely sensitive to free phosphate. If the sample contains phosphate, remove it by at least 3 washes with a 10 kDa NMWL membrane filter. |
| 2 | Prepare the Phytic Acid Working Reagent (75 µL per well) by mixing 1 µL of the Phytic Acid stock with 80 µL of Assay Buffer per assay well. Prepare the Colour Development Reagent (20 µL per well) by mixing 100 volumes of POMG Reagent A with 1 volume of POMG Reagent B. Bring all reagents to room temperature before use and check that enzyme preparations and buffers are free of phosphate. |
| 3 | Pipette 5 µL of phytase-containing extract into separate wells of a clear-bottom 96-well plate, reserving one well as a Blank (5 µL buffer, no enzyme). Initiate the reaction by adding 75 µL of Working Reagent to each well and tap briefly to mix. |
| 4 | Incubate the plate at room temperature (or the desired temperature) for 30 minutes. |
| 5 | Phosphate standards: prepare a 40 µM phosphate premix by adding 20 µL of the 1 mM phosphate standard to 480 µL of distilled water, then dilute to give standards of 40, 24, 12 and 0 µM. Pipette 80 µL of each standard in duplicate into separate wells. |
| 6 | At 30 minutes, add 20 µL of the Colour Development Reagent to each well and mix gently by tapping. |
| 7 | Incubate for a further 30 minutes at room temperature for colour development. |
| 8 | Measure absorbance at 600-660 nm (peak 620 nm) with a plate reader. If the observed phosphate concentration equals or exceeds the 40 µM standard, dilute the enzyme extract in buffer and repeat. |
Phytase Activity = ((ODSAMPLE - ODBLANK) / Slope) x Reaction Volume (L) / time / Sample Volume (L) = ((ODSAMPLE - ODBLANK) / Slope) x 0.533 (U/L), where Slope is the slope (per µM) of the phosphate standard curve. One unit (U) of enzyme catalyses the release of 1 µmole of substrate per minute under the assay conditions (pH 5.5).
| Component | Quantity | Storage |
| Assay Buffer (pH 5.5) | 10 mL | 4°C |
| Phytic Acid | 120 µL | 4°C |
| Standard (1 mM Phosphate) | 120 µL | 4°C |
| POMG Reagent A | 2.5 mL | 4°C |
| POMG Reagent B | 120 µL | 4°C |
