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 4ยฐC 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 ยตL per well) by mixing 1 ยตL of the Phytic Acid stock with 80 ยตL of Assay Buffer per assay well. Prepare the Colour Development Reagent (20 ยตL 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 ยตL of phytase-containing extract into separate wells of a clear-bottom 96-well plate, reserving one well as a Blank (5 ยตL buffer, no enzyme). Initiate the reaction by adding 75 ยตL 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 ยตM phosphate premix by adding 20 ยตL of the 1 mM phosphate standard to 480 ยตL of distilled water, then dilute to give standards of 40, 24, 12 and 0 ยตM. Pipette 80 ยตL of each standard in duplicate into separate wells. |
| 6 | At 30 minutes, add 20 ยตL 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 ยตM 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 ยตM) of the phosphate standard curve. One unit (U) of enzyme catalyses the release of 1 ยตmole of substrate per minute under the assay conditions (pH 5.5).
| Component | Quantity | Storage |
| Assay Buffer (pH 5.5) | 10 mL | 4ยฐC |
| Phytic Acid | 120 ยตL | 4ยฐC |
| Standard (1 mM Phosphate) | 120 ยตL | 4ยฐC |
| POMG Reagent A | 2.5 mL | 4ยฐC |
| POMG Reagent B | 120 ยตL | 4ยฐC |
