Invitrogen™ Amplex™ Red Sphingomyelinase Assay Kit

Catalog No.	Quantity
A12220	500 Assays

Catalog No. A12220 Supplier Invitrogen™ Supplier No. A12220

Description

Detects very low concentrations of sphingomyelinase

The Amplex Red Sphingomyelinase Assay Kit provides a sensitive, rapid, and simple fluorometric method for detecting very low concentrations of sphingomyelinase using a fluorescence microplate reader or fluorometer. In this enzyme-coupled assay, sphingomyelinase activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red reagent), a sensitive fluorogenic probe for hydrogen peroxide. Sphingomyelinase hydrolyses the sphingomyelin to yield ceramide and phosphorylcholine. Alkaline phosphatase is added, which hydrolyzes phosphorylcholine to form choline. The 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 sphingomyelinase activity levels as low as 80μU/mL in a 200μL assay volume
• 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: Wet ice

Specifications

• Content And Storage: Store in freezer -5°C to -30°C and protect from light.
• Detection Method: Fluorescence
• For Use With (Application): Sphingomyelinase Assay
• For Use With (Equipment): Fluorometer, Microplate Reader
• Product Type: Amplex Red Assay Kit
• Dye Type: Other Label(s) or Dye(s)
• Format: 96-well plate
• Product Line: Amplex
• Quantity: 500 Assays
• Shipping Condition: Wet Ice

Frequently Asked Questions (FAQs)

I'm using an Amplex Red kit, the reagent changes color to pink almost immediately in my own Krebs-Ringer buffer but not in HBSS. Why is this?

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.

Can Amplex Red Assays be performed using cell lysates?

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.