Invitrogen™ PiPer™ Pyrophosphate or Phosphate Assay Kit 

Description

PiPer Pyrophosphate and Phosphate Assay Kit provides an ultrasensitive assay to detect the fluorescent product resorufin. These kits can be used to detect inorganic pyrophosphate (PPi) or phosphate (Pi) in a variety of samples or can monitor the kinetics of their release by a variety of enzymes, including DNA/RNA polymerases, adenylate cyclase, S-acetyl coenzyme A synthetase, ATPases, GTPases, 5′-nucleotidase, protein phosphatases, acid/alkaline phosphatases and phosphorylase kinase. As resorufin has a strong absorption, this assay can be performed either fluorometrically or colorimetrically.

Key features include:

• High sensitivity—detects low levels of inorganic phosphate
• Versatility—suitable for a wide range of applications including enzyme activity assays
• Quantitative results—provide accurate and reproducible results
• Number of labelings per kit: ~1,000 (100 μL per assay volume)
• Detection method: colorimetric, fluorescence
• Compatibility: can be used with both microplate readers and spectrophotometers
• Excitation/emission maxima: 563/587 nm

The PiPer Pyrophosphate Assay Kit provides a sensitive fluorogenic or colorimetric method for detecting as little as 0.4 μM (fluorescence) or 2 μM (absorbance) inorganic pyrophosphate in a 100 μL assay volume. The Phosphate Assay Kit detects as little as 0.2 μM Pi (fluorescence) in purified enzyme systems with a 100 μL assay volume.

Use the PiPer Pyrophosphate and Phosphate assay kits for applications such as anion detection, biochemical toxicology assays, cell analysis, cell metabolism, cell viability, cellular toxicology assays, drug discovery and development, enzyme and protein activity assays, ionic homeostasis and signaling, phosphatase activity, target and lead identification and validation.

The absorption and fluorescence of resorufin are pH dependent. There is a pKa at ~6.0 where the absorption maximum shifts to ~480 nm and the fluorescence quantum yield is markedly lower. Given Amplex™ Red reagent is unstable at high pH (>8.5) and is sensitive to light, we recommend this reaction be performed using the provided reaction buffer (at optimal pH ~7.5) for best results and reagents kept protected from light.

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