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Fluorimetric detection of ATPase and protein phosphatase activities by monitoring formation of inorganic phosphate

by Jianjun He, Zhenjun Diwu, Jinfang Liao

Introduction


Cells utilize a wide variety of phosphate and polyphosphate esters as enzyme substrates, second messengers, membrane structural components and vital energy reservoirs. Phosphate is involved in many biological processes. Phosphatases, ATPases and several other enzymes catalyze biochemical reactions in which inorganic phosphate (Pi) is released from a phosphoester substrate. Detection of many phosphoester–metabolizing enzymes is difficult because suitable substrates are not available. We have developed a fluorimetric Pi detection system that can be used for detecting activity of any Pi-generating enzymes. The assay provides sensitive detection of phosphate, an alternative to hazardous radioactive methods and other less sensitive colorimetric assays. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation with no separation steps required.

ATPase or phosphatase activity is determined by the monitoring of Pi formation by Pi sensors. ABD Bioquest offers three Pi sensors that can be used at the following wavelengths:

  • Pi Sensor Red: Ex/Em = 540 nm/590 nm (pH-sensitive)
  • Pi Sensor UltraRed: Ex/Em = 545 nm/596 nm (pH-insensitive)
  • Pi Sensor NIR: Ex/Em = 645 nm/670 nm (pH-insensitive)

 

Materials and Methods


  1. All Standard dilutions and phosphatases reactions were performed at room temperature with PhosphoWorks™ Fluorimetric Phosphate Assay Kit.
  2. Standard curves and Phosphatase reactions were performed in 20 µl volumes in costar 384-solid black well plates for 30 min.
  3. Phosphate was measured as described in PhosphoWorks™ Fluorimetric Phosphate Assay Kit by using a NOVOstar microplate reader (BMG LabTech) at EX 540/Em 590.

 

Results


Figure1.  Phosphate dose response on 96-well black plate using a BMG Novostar microplate reader (BMG Labtech) measured with the PhosphoWorks™ Fluorimetric Phosphate Assay Kit. As low as 0.1 µM phosphate can be detected with 1 h incubation time.

Figure 2.  Alkaline phosphatase detection with the PhosphoWorks™ Fluorimetric Phosphate Assay Kit. The phosphatase was incubated in the presence of PNPP for 30 minutes at room temperature, and phosphate generation was detected at 10 min incubation time using the PhosphoWorks™ Fluorimetric Phosphate Assay Kit.

Figure 3.  Acid phosphatase detection with the PhosphoWorks™ Fluorimetric Phosphate Assay Kit. The phosphatase was incubated in the presence of PNPP for 30 minutes at room temperature, and phosphate generation was detected at 10 min incubation time using the PhosphoWorks™ Fluorimetric Phosphate Assay Kit.

Figure 4.  ATPase detection with the PhosphoWorks™ Fluorimetric Phosphate Assay Kit. The ATPase was incubated in the presence of ATP for 30 minutes at room temperature, and phosphate generation was detected at 10 min incubation time using the PhosphoWorks™ Fluorimetric Phosphate Assay Kit.

 

Conclusions


  1. PhosphoWorks™ Fluorimetric Phosphate Assay is a sensitive, fast, simple, and homogeneous assay for measuring phosphatases and ATPases activities.
  2. As low as 0.1 µM phosphate can be detected.
  3. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation with no separation steps required.


Original created on April 29, 2007, last updated on April 29, 2007
Tagged under: Measuring Phosphotase Activity