Lipases and inorganic phosphates are critical in many signaling pathways, from mTOR to GPCRs where they are involved in disease states ranging from cardiovascular to inflammation and cancer.

Measuring phosphates and phosphatase activity in solution is fundamental to an understanding of cellular dynamics in multiple models. Molecular Probes® offers a series of sensitive assays with fluorescent readout to address a diverse assortment of analytes.  

Cholesterol

The Amplex® Red Cholesterol Assay Kit provides a sensitive, rapid, and simple fluorometric method for detecting very low concentrations of cholesterol using a fluorescence microplate reader or fluorometer. The assay measures both free cholesterol and cholesteryl esters and allows for multiple time point measurements.

Assays for Phosphate and Pyrophosphate

PiPer™ Kits provide ultra-sensitive one-step methods for the detection of free phosphate or pyrophosphate in solution. The kits can be used with a variety of sample types to measure the kinetics of phosphate or pyrophosphate release by enzymes including ATP-ases, GTP-ases, protein phosphatases and kinases. Signal is reported using an enzyme-coupled reaction with Amplex® Red.

Phosphatase Assays

EnzCheck® Assay Kits are designed to detect low levels of phosphatase activity in purified or complex systems.  They are offered as reagents with buffers and controls or pre-dispensed into 96-well or 384-well microplates.  Enzyme selectivity is achieved by using the inhibitors included in the kit.

Phospholipase Assays

Amplex® Red Phospholipase Kits offer a sensitive method for continuously monitoring phospholipase activity in vitro using a fluorescence microplate reader. The Amplex® Red reagent generates a highly fluorescent signal using an enzyme-coupled reaction for exquisite sensitivity.

Amplex® Red Cholesterol Assay Kit
Detection of cholesterol using the Amplex® Red Cholesterol Assay Kit. Each reaction contained 150 µM Amplex® Red reagent, 1 U/mL HRP, 1 U/mL cholesterol oxidase, 1 U/mL cholesterol esterase, and the indicated amount of cholesterol in 1X reaction buffer. Reactions were incubated at 37°C for 30 minutes. Fluorescence was measured with a fluorescence microplate reader using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. The inset shows the high sensitivity and excellent linearity of the assay at low levels of cholesterol (0–0.015 µg/mL).

Selection guides
  Amplex® Red Cholesterol Assay Kit
Target Cholesterol
Reporter Amplex® Red reagent
Ex/Em (nm) 571/585
Live cell No
Lysate Yes
Purified enzyme Yes
Usage Lower detection limit 200 nM
Components Includes assay buffers and controls
Format 500 assays using 200 µL reaction volume
Protocol outline
  1. Prepare standard curve samples, load into wells.
  2. Dilute experimental samples in reaction buffer, load into wells.
  3. Prepare Amplex® Red working solution.
  4. Add Amplex® Red solution to wells.
  5. Incubate.
  6. Measure fluorescence.
Cat. No. A12216
  PiPer™ Phosphate Assay Kit PiPer™ Pyrophosphate Assay Kit
Target Phosphate Pyrophosphate
Reporter Amplex® Red reagent Amplex® Red reagent
Ex/Em (nm) 571/585 571/585
Live cell No No
Lysate Yes Yes
Purified enzyme Yes Yes
Usage Lower detection limit 0.2 µM phosphate Lower detection limit 0.4 µM pyrophosphate
Components Includes assay buffers and controls Includes assay buffers and controls
Format 1,000 assays using 100 µL reaction volume 1,000 assays using 100 µL reaction volume
Protocol outline
  1. Prepare standard curve samples, load into wells.
  2. Dilute experimental samples in reaction buffer, load into wells.
  3. Prepare Amplex® Red working solution.
  4. Add Amplex® Red solution assay wells.
  5. Incubate.
  6. Measure fluorescence
Cat. No. P22061 P22062
  EnzChek® Phosphatase Assay Kit RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit RediPlate™ 96 EnzChek® Tyrosine Phosphatase Assay Kit
Target Acid phosphatase Serine/Threonine phosphatase Tyrosine phosphatase
Reporter DiFMUP DiFMUP DiFMUP
Ex/Em (nm) 358/455 358/455 358/455
Live cell No No No
Lysate Yes Yes Yes
Purified enzyme Yes Yes Yes
Usage ~2 µM Pi (~0.2 µg/mL) ~2 µM Pi (~0.2 µg/mL) ~2 µM Pi (~0.2 µg/mL)
Components Includes assay buffers and controls Includes assay buffers, reference standards and inhibitors ensuring activity only from Ser/Thr phosphatase are monitored Includes assay buffers, reference standards and inhibitors ensuring activity only from tyrosine phosphatases are monitored
Format 1,000 assays using 100 µL reaction volume 96 assays pre-dispensed into microplate wells
Protocol outline
  1. Prepare the DiFMUP substrate, add to wells.
  2. Add the samples and controls to wells.
  3. Incubate the samples.
  4. Measure fluorescence.
  5. Determine the phosphatase activity using the standard curve.
  1. Optimize reaction buffer by adding appropriate additive(s).
  2. Rehydrate substrate and reference standards.
  3. Add samples to assay wells.
  4. Incubate.
  5. Measure fluorescence.
  6. Determine the serine/threonine phosphatase activity using standard curve
Cat. No. E12020 R33700 R22067
  Amplex® Red Phosphatidylcholine-Specific Phospholipase C Assay Kit Amplex® Red Phospholipase D Assay Kit
Target Phospholipase C Phospholipase D
Reporter Amplex® Red Amplex® Red
Ex/Em (nm) 571/585 571/585
Live cell No No
Lysate Yes Yes
Purified enzyme Yes Yes
Usage Lower detection limit 2 x 10–4 U/mL Lower detection limit 1 x 10–2 U/mL
Components Includes assay buffers and controls Includes assay buffers and controls
Format 500 assays using 200 µL reaction volume 500 assays using 200 µL reaction volume
Protocol outline
  1. Prepare standard curve samples, add to wells.
  2. Dilute experimental samples in reaction buffer, add to wells.
  3. Prepare Amplex® Red working solution.
  4. Add Amplex® Red solution to wells.
  5. Incubate.
  6. Measure fluorescence.
  1. Prepare standard curve samples, add to wells.
  2. Dilute experimental samples in reaction buffer, add to wells.
  3. Prepare Amplex® Red working solution.
  4. Add Amplex® Red solution to wells.
  5. Incubate.
  6. Measure fluorescence.
Cat. No. A12218 A12219
For Research Use Only. Not for use in diagnostic procedures.