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Invitrogen™ Amplex™ Red Phosphatidylcholine-Specific Phospholipase C Assay Kit
Description
The Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit provides a sensitive method for detecting phosphatidylcholine-specific phospholipase C (PC-PLC) activity using a fluorescence microplate reader or fluorometer. In this enzyme-coupled assay, PC-PLC activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red reagent), a sensitive fluorogenic probe for hydrogen peroxide. PC-PLC converts phosphatidylcholine (lecithin) to phosphocholine and diacylglycerol. Alkaline phosphatase is added, which hydrolyzes phosphorylcholine to form choline. Choline is then oxidized by choline oxidase to form betaine and hydrogen peroxide. In the presence of horseradish peroxidase, the hydrogen peroxide reacts with Amplex Red reagent in a 1:1 stoichiometric ratio to generate the highly fluorescent product resorufin. Because resorufin has absorption and fluorescence emission maxima of approximately 571nm and 585nm, respectively, there is little interference from autofluorescence in most biological samples.
- Detects phospholipase-C activity levels as low as 0.2mU/mL
- Format allows for multiple time point measurements
- Designed for minimal autofluorescence interference
- Custom assay design and packaging are also available
Use Amplex Red Assays for a Broad Range of Investigations
A wide variety of validated Amplex Red assays are available for studying cell signaling and lipids, neurobiology, inflammation and immune function, and metabolism. We also offer Amplex UltraRed Reagent, a second-generation reagent providing greater sensitivity and brighter fluorescence, and the Amplex Red/UltraRed Stop Reagent. The Amplex Red/UltraRed Stop Reagent provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After addition of the stop reagent, the fluorescence signal remains stable for at least three hours.
Enzyme and Protein Activity Assays, Lipase and Phopholipase Activity, Proteins, Expression, Isolation and Analysis
Order Info
Shipping Condition: Room temperature
Specifications
Specifications
| Content And Storage | Store in freezer -5°C to -30°C and protect from light. |
| Detection Method | Fluorescence |
| For Use With (Application) | Phospholipase Assay |
| For Use With (Equipment) | Fluorometer, Microplate Reader |
| Product Type | Amplex Red Assay Kit |
| Dye Type | Other Label(s) or Dye(s) |
| Format | Cuvettes, 96-well plate |
| Product Line | Amplex |
| Quantity | 500 Assays |
| Shipping Condition | Room Temperature |
Frequently Asked Questions (FAQs)
The components of Krebs-Ringer buffer (salts) should not cause oxidation of the Amplex reagent (which, in the presence of peroxidase and H2O2 oxidizes to resorufin, which is pink in color and fluorescent). Try water alone (the water used to make the Krebs-Ringer buffer). Since Hank's Buffered Saline Solution is typically purchased rather than made in the lab, it likely would not have the same contaminant. Another option is to degas the buffer prior to use to removed dissolved oxygen radicals.
This is not recommended. The presence of endogenous proteases can complicate the assay by degrading the horseradish peroxidase (HRP). Endogenous peroxidases and antioxidants can modify the H2O2 required for the reaction, competing with HRP (and catalase) for the substrate.
The Amplex Red Assays are best performed with either purified enzymes or extracted H2O2 in a defined buffer system, extracellular solutions or body fluids (media, serum, etc.) that do not exhibit high levels of endogenous protease or oxidase activity and do not contain antioxidants.
For Research Use Only. Not for use in diagnostic procedures.