| Field | Specification |
|---|---|
| Mfr No | |
| Assay Time | |
| Detection Method | |
| Product Type | |
| Sample Type(s) | Enzyme reactions with free phosphate releases |
| Shipping | |
| Species | |
| Storage |
Overview
For sensitive and high-throughput phosphate determination. The assay uses OD620nm (malachite green) for signal readout. Compatible sample input includes Enzyme reactions with free phosphate releases. Typical stated assay timing is 30 min.
Key elements and design rationale
- Readout format: OD620nm (malachite green) supports plate-based signal acquisition and consistent comparison across matched samples.
- Sample compatibility: The stated sample scope includes Enzyme reactions with free phosphate releases, which is useful when aligning matrix type with calibration and control design.
- Analytical range context: The supplied specifications include a stated detection limit of 0.4 µM for interpreting low-signal samples.
- Feature emphasis: Reagent very stable. Due to our innovative formulation, no precipitation of reagent occurs. Therefore no filtration of reagent is needed prior to assays, as is often required with other commercial kits.
Additional feature notes highlight High sensitivity and wide detection range: detection of as little of 20 pmoles of phosphate and useful range between 0.4 µM and 50 µM phosphate; Fast and convenient: single reagent “mix-and-measure” assay allows quantitation of free phosphate within 30 minutes. Available format information for this listing includes 500 Tests.
Biological background
This product is centered on measurement of piblue phosphate within the matrices described for the assay. In practice, datasets from this type of format are typically interpreted by comparing relative signal, activity, or abundance across matched control and experimental groups rather than relying on a single value in isolation. Careful alignment of sample matrix, incubation window, and calibration strategy is important when comparing results across plates, operators, or study days.
More details
The PiBlue™ Phosphate Assay Kit is based on a proprietary formulation of the malachite green dye. The PiBlue™ reagent forms a blue colored complex with free orthophosphate. The rapid color formation from the reaction can be conveniently measured on a spectrophotometer (600 – 660 nm) or on a plate reader. The non-radioactive colorimetric assay kits have been optimized to offer superior sensitivity and prolonged shelf life. The assay is simple and fast, involving a single addition step for phosphate determination. Assays can be performed in tubes, cuvettes or multi-well plates. The assays can be conveniently executed in 96-well plates for high-throughput screening of enzyme inhibitors.
Detection method
Colorimetric (OD 620 nm).
Detection limit and analytical sensitivity
Reported detection limit: 0.4 µM.
Procedures and timing
Stated procedure or timing information: 30 min.
Research relevance and current trends
- Plate-based quantification and side-by-side group comparison remain central use cases for this assay format.
- The product notes emphasize multi-sample throughput, making it relevant for screening-oriented and larger batch comparison studies.
- Short assay timing and plate compatibility support time-course or repeated-measure collection plans when handling is kept consistent.
Common research applications
- Quantify piblue phosphate in enzyme reactions with free phosphate releases by OD620 nm (malachite green) readout.
- Compare treatment or phenotype groups using matched enzyme reactions with free phosphate releases handling.
- Monitor time-course or pre/post changes in enzyme reactions with free phosphate releases across study conditions.
Interpretation is usually strongest when signal changes are assessed alongside matrix-matched controls, replicate agreement, and the assay's stated analytical window.
Notes for experimental interpretation
- Matrix composition, background signal, and sample handling can influence apparent response; compare like-with-like whenever possible.
- Use appropriate blanks, controls, and replicate wells to distinguish biological differences from plate, reagent, or handling variability.
What is the difference between your 3 phosphate assays: DIPI-500, POMG-25H, POPB-500?
All three products are based on the malachite green dye. While the PiBlue (POPB-500) and QuantiChrom (DIPI-500) Phosphate Assay Reagents are identical, they differ in the standards. The PiBlue is designed for customers who want prepare a full calibration curve for their assays, and the latter has one set of a blank control and 30 µM phosphate standard. The reagent is more diluted than the reagent used in the Malachite Green Phosphate Assay Kit (POMG-25H). As a result, one adds 100 µL reagent (POPB-500 or DIPI-500) to 50 µL sample, whereas for the Malachite Green based assay, one adds 20 µL Reagent to 80 µL sample.
In practice, the DIPI-500 is the most convenient kit to use, because there is no need to prepare a calibration curve there is no need for extra mixing. Because adding a larger volume (100 μL) to a small volume (50 μL) effects efficient mixing in well, no extra mixing is needed. The POPB-500 requires preparation of calibration curve. With the POMG-25H Kit, one needs to mix the reagent with the sample, because adding 20 µL to 80 µL does not mix efficiently by itself. Also a preparation of a standard curve is necessary.
I’m using the Malachite Green Phosphate Assay Kit (POPB-500) to detect GTPase activity. However, I saw high background and no increase in phosphate liberation.
This assay is very sensitive to free phosphate. Before each assay, it is important to check that all materials (substrates, enzyme preparations and buffers) are pure and free of contaminating phosphate. This can be conveniently done by adding 20 µL of the Working Reagent to 80 µL sample solution. The blank OD values at 620 nm should be lower than 0.2. If the OD readings are higher than 0.2, check water phosphate level. Double distilled water usually have OD readings lower than 0.1. Lab detergents may contain high levels of phosphate. Make sure that lab wares are free from contaminating phosphate after thorough washes.
In the case of enzyme reactions, the reaction may be terminated directly by the addition of the acidic Working Reagent. Dilution of reaction mixture may be necessary prior to the assay. For ATPase or GTPase assays, the ATP or GTP concentration should be lower than 0.25 mM. If the reaction mixture contains > 0.25mM ATP or GTP, dilute samples in distilled water. For example, if the ATPase reaction contained 1 mM ATP, at the end of reaction dilute reaction mixture 4-fold in water prior to the assay.
The chromogenic reaction is completed within 30 min at room temperature. Read OD at or shorter than 30 min to minimize background due to slow hydrolysis of the substrates (ATP or GTP) by the acidic reagent.
If the background OD is in the normal range, but there is still no free phosphate detected, it is likely the GTPase was not active. To confirm this possibility, one could run a test without the enzyme by spiking free phosphate (e.g. 0.5 – 40 µM) into the mixture of Buffer and Substrate. In this case, one should see an increase in OD with increasing free phosphate concentrations. This confirms that the enzyme was not active in the assay.
For laboratories requiring additional technical capacity, we provide scientific support services including assay execution, method guidance, product sourcing, and customization to align the assay with specific experimental objectives. If you need assistance selecting the appropriate kit configuration, adapting the workflow to your application, or identifying related research services, please click Talk to a Scientist, email support@biohippo.com, or review our Research Services; a member of our scientific team will follow up with recommendations tailored to your study.
In vitro characterization of a novel consensus bacterial 6-phytase and one of its variants
Christensen, T., et al. (2020). In vitro characterization of a novel consensus bacterial 6-phytase and one of its variants. Current Biochemical Engineering 6(3): 156-171. Assay: Phosphate in Trichoderma reesei phytase.
Investigation of the effects of actinorhodin biosynthetic gene cluster expression and a rpoB point mutation on the metabolome of Streptomyces coelicolor M1146
Nitta, K., et al. (2021). Investigation of the effects of actinorhodin biosynthetic gene cluster expression and a rpoB point mutation on the metabolome of Streptomyces coelicolor M1146. Journal of Bioscience and Bioengineering. Assay: Phosphate in Streptomyces coelicolor M1146 cells.
Multi-omics analysis of the effect of cAMP on actinorhodin production in Streptomyces coelicolor
Nitta, K., et al. (2020). Multi-omics analysis of the effect of cAMP on actinorhodin production in Streptomyces coelicolor. Frontiers in Bioengineering and Biotechnology 8: 595552. Assay: Phosphate in bacteria nutrition medium.
Impact of phosphate availability on membrane lipid content of the model strains, Streptomyces lividans and Streptomyces coelicolor
Lejeune, C., et al. (2021). Impact of phosphate availability on membrane lipid content of the model strains, Streptomyces lividans and Streptomyces coelicolor. Frontiers in Microbiology 12: 623919. Assay: Phosphate in R2YE agar medium.
Delivery of Formulated Industrial Enzymes with Acoustic Technology
Hwang, J. D., Ortiz-Maldonado, M., & Paramonov, S. (2016). Delivery of Formulated Industrial Enzymes with Acoustic Technology. Journal of laboratory automation, 21(1), 153-165. Assay: Phosphate in Buttiauxella sp Phytase.
Effects of viscogens on RNA transcription inside reovirus particles
Demidenko AA et al (2011). Effects of viscogens on RNA transcription inside reovirus particles. J Biol Chem. 286(34):29521-30. Assay: Phosphate in rat lipid.
Competition between foliar Neotyphodium lolii endophytes and mycorrhizal Glomus spp
Liu Q et al (2011). Competition between foliar Neotyphodium lolii endophytes and mycorrhizal Glomus spp. fungi in Lolium perenne depends on resource supply and host carbohydrate content. Functional Ecology 25:910-920. Assay: Phosphate in mouse intracellular phosphate.
Fenretinide promotes functional recovery and tissue protection after spinal cord contusion injury in mice
Lopez-Vales R, et al (2010). Fenretinide promotes functional recovery and tissue protection after spinal cord contusion injury in mice. J Neurosci. 30(9):3220-6. Assay: Phosphate in mouse lipid.
Increased lipid peroxidation and abnormal fatty acid profiles in seminal and blood plasma of normozoospermic males from infertile couples
Oborna I et al (2010). Increased lipid peroxidation and abnormal fatty acid profiles in seminal and blood plasma of normozoospermic males from infertile couples. Hum Reprod. 25(2):308-16. Assay: Phosphate in fish marine teleost follicles, eggs.
Increased lipid peroxidation and abnormal fatty acid profiles in seminal and blood plasma of normozoospermic males from infertile couples
Oborna I et al. (2010). Increased lipid peroxidation and abnormal fatty acid profiles in seminal and blood plasma of normozoospermic males from infertile couples. Hum Reprod. 25(2):308-16. Assay: Phosphate in human blood, semen.
Inorganic pyrophosphatase induces type I collagen in osteoblasts
Polewski MD, et al (2010). Inorganic pyrophosphatase induces type I collagen in osteoblasts. Bone 46(1):81-90. Assay: Phosphate in mouse/human cellular extract.
Ups and downs of intestinal function with prolonged fasting during aestivation in the burrowing frog, Cyclorana alboguttata
Cramp RL et al (2009). Ups and downs of intestinal function with prolonged fasting during aestivation in the burrowing frog, Cyclorana alboguttata. J Exp Biol. 212(22):3656-63. Assay: Phosphate in mouse lipid.
Ups and downs of intestinal function with prolonged fasting during aestivation in the burrowing frog, Cyclorana alboguttata
Cramp RL, et al (2009). Ups and downs of intestinal function with prolonged fasting during aestivation in the burrowing frog, Cyclorana alboguttata. J Exp Biol. 212(22):3656-63. Assay: Phosphate in frog Na+/K+-ATPase extract.
Tripartite purinergic modulation of central respiratory networks during perinatal development: the influence of ATP, ectonucleotidases, and ATP metabolites
Huxtable AG, et al (2009). Tripartite purinergic modulation of central respiratory networks during perinatal development: the influence of ATP, ectonucleotidases, and ATP metabolites. JNeurosci. 29(47):14713-25. Assay: Phosphate in rat ATPase extract.
Altered TNSALP expression and phosphate regulation contribute to reduced mineralization in mice lacking androgen receptor
Kang HY et al (2008). Altered TNSALP expression and phosphate regulation contribute to reduced mineralization in mice lacking androgen receptor. Mol Cell Biol.28(24):7354-67. Assay: Phosphate in rat ATPase extract.
Altered TNSALP expression and phosphate regulation contribute to reduced mineralization in mice lacking androgen receptor
Kang HY, et al (2008). Altered TNSALP expression and phosphate regulation contribute to reduced mineralization in mice lacking androgen receptor. Mol Cell Biol.28(24):7354-67. Assay: Phosphate in mouse cytosolic fraction.
The isolation and structure of membrane lipid rafts from rat brain
Chen X et al (2007). The isolation and structure of membrane lipid rafts from rat brain. Biochimie 89(2):192-6. Assay: Phosphate in mouse/human cellular extract.
The isolation and structure of membrane lipid rafts from rat brain
Chen X, et al (2007). The isolation and structure of membrane lipid rafts from rat brain. Biochimie 89(2):192-6. Assay: Phosphate in rat lipid.
Novel mouse model of autosomal semidominant adult ypophosphatasia has a splice site mutation in the tissue nonspecific alkaline phosphatase gene Akp2
Hough TA et al (2007). Novel mouse model of autosomal semidominant adult ypophosphatasia has a splice site mutation in the tissue nonspecific alkaline phosphatase gene Akp2. J Bone Miner Res. 22(9):1397-407. Assay: Phosphate in frog Na+/K+-ATPase extract.
Novel mouse model of autosomal semidominant adult hypophosphatasia has a splice site mutation in the tissue nonspecific alkaline phosphatase gene Akp2
Hough TA, et al (2007). Novel mouse model of autosomal semidominant adult hypophosphatasia has a splice site mutation in the tissue nonspecific alkaline phosphatase gene Akp2. J Bone Miner Res. 22(9):1397-407. Assay: Phosphate in mouse intracellular phosphate.
An osteoclastic protein-tyrosine phosphatase is a potential positive regulator of the c-Src protein-tyrosine kinase activity: a mediator of osteoclast activity
Lau K.H. et al. (2006). An osteoclastic protein-tyrosine phosphatase is a potential positive regulator of the c-Src protein-tyrosine kinase activity: a mediator of osteoclast activity. J Cell Biochem. 97(5):940-55. Assay: Phosphate in rabbit phosphatases.
