Dye-based detection of inorganic phosphate
August 25th, 2017
Dyes such as Malachite Green are regularly used to detect inorganic phosphate (Pi), an important inorganic ion in a wide variety of biological functions. In the presence of Pi these reagents change color, and a simple absorbance reading can be employed to measure the amount of Pi which has been released from a substrate.
Principle of dye-based detection of inorganic phosphate. Enzyme activity results in the release of Pi from the enzyme substrate. The reaction is halted by the addition of the dye reagent (orange), which turns green in the presence of Pi.
Since these dyes are universal, rather than being enzyme-specific, they can be used to measure the activity of any enzyme which generates Pi. They are commonly employed during drug discovery programmes which aim to identify specific enzyme inhibitors.
|Common enzyme classes which generate Pi|
|GTPases||Nucleases / helicases|
|Gyrases / topoisomerases||Heat shock proteins|
|Pyrophosphate-generating enzymes e.g. adenylate cyclase||Monophosphate substrates of PNPP e.g. inositol monophosphatase|
|P-glycoprotein||Polynucleotide kinase/phosphatase (PNKP)|
PiColorLock™ – a superior phosphate detection reagent
Unfortunately, commercially available dyes suffer from two major problems 1) the dye-Pi complexes are frequently prone to precipitation, preventing the sample from being read successfully, and 2) the dye reagents are acidic, which can lead to non-enzymatic hydrolysis of many phosphorylated substrates, resulting in background signal. To circumvent these issues, we developed our PiColorLock™ Phosphate Detection System. The unique formulation of this reagent makes it superior to many competitor products, and offers several key advantages:
- Accelerator to speed up color development
- Stabilizer to suppress non-enzymatic backgrounds with acid-labile substrates such as ATP / GTP
- Enhanced assay linearity
- Wide dynamic range
- Color development is not inhibited by high concentrations of protein
- Compatible with almost any assay buffer, including DMSO
- PiColorLock™-Pi complex is very stable
Avoid reagent precipitation
PiColorLock™ contains special additives to enhance the stability of dye complexes. These reagents eliminate the formation of precipitates, which can prevent the sample from being read successfully. They also allow for samples to be read much later than those prepared using competitor products, making PiColorLock™ ideal for High Throughput Screening (HTS) formats.
PiColorLock™-Pi complexes are highly stable. Many competitor reagents form unstable dye complexes, requiring samples to be read promptly. The image on the left shows the effect of adding Pi to three commercial dye reagents and incubating for 1 hour; precipitation is seen with the competitor products but not for PiColorLock™. The image on the right clearly demonstrates the stability of the absorbance reading over an extended period.
Avoid non-enzymatic decay
PiColorLock™ contains a proprietary stabilizer to suppress non-enzymatic background signal. This prevents non-enzymatic hydrolysis of acid-labile substrates such as ATP and GTP, allowing a low background signal to be maintained.
PiColorLock™ contains a propietary stabilizer to suppress non-enzymatic background signal. Many competitor reagents are prone to high background signal, which occurs due to the breakdown of acid-labile substrates. The image on the left illustrates the effect of incubating ATP with three commercial dye-based detection reagents; the competitor products demonstrate increasing background over time, while PiColorLock™ maintains negligible backgrouhd signal. Please note that PiColorLock™ Gold has been superseded by PiColorLock™. The image on the right shows the effect of omitting the stabilizer solution.
Enhance assay linearity
PiColorLock™ provides a significantly larger linear range than many competitor products, affording greater assay flexibility.
PiColorLock™ affords an enhanced linear range. Many competitor reagents have a narrow linear range; for example, the maximum signal that can be generated with competitor A is just 0.5 absorbance units. Please note that PiColorLock™ ALS has been superseded by PiColorLock™.
The PiColorLock™ product range
PiColorLock™ is at the core of our phosphate detection product range, which also includes our ATPase assay kits and GTPase assay kits, for measuring the activity of ATPases and GTPases respectively. The lyophilized 10mM ATP/GTP included with these kits is ultra-high quality to ensure the lowest possible assay background; these vials are also available for separate purchase.
|Kit component||PiColorLock™ Phosphate Detection System||High Throughput Colorimetric ATPase Assays||High Throughput Colorimetric GTPase Assays|
Kit components within the PiColorLock™ product range.
PiBind™ resin – quick and easy removal of Pi contamination
Many substrate buffers contain free Pi, which should be removed to prevent background signal in the downstream assay. While dialysis is a relatively simple method of Pi removal, it is a rather slow process; desalting, although quicker, can be difficult to perform and is limited to small volumes.
Our PiBind™ resin was developed to provide a user-friendly solution to the removal of Pi from buffers. In most applications, the PiBind™ resin is simply added to the contaminated sample or buffer solution, which is then gently stirred or agitated; after adsorption of Pi, the resin is removed by centrifugation or filtration.
PiBind™ resin is effective over a broad range of pH values. Removal of 1mM Pi from buffers of varying pH; PiBind™ is unaffected by many commonly used buffer additives.
References and resources
The use of PiColorLock™ has been cited in almost 200 publications to date, with the reagent being employed within a vast diversity of research areas.
Download our free guide to learn more about PiColorLock™, or watch our webinar for an overview of drug screening assays with a particular focus on phosphate-generating enzymes.
For further information about any of our products, please contact us.