Invitrogen™ Amplex™ Red and Amplex™ UltraRed Reagents

Description

• The Amplex Red and Amplex UltraRed reagents are highly sensitive and stable fluorescent probes for the detection of hydrogen peroxide (H 2 O 2 ) and ideal fluorogenic substrates for peroxidase
• These reagents can be used for solution-based assays to detect the activity of H 2 O 2 released from biological samples or with HRP-based ELISA applications
• The Amplex UltraRed reagent is an improved version of Amplex Red that is brighter and more sensitive to peroxidase with less pH sensitivity compared to the Amplex Red reagent
• In the presence of horseradish peroxidase (HRP), the Amplex Red and UltraRed reagents react in a 1:1 stoichiometry with H 2 O 2 to produce highly fluorescent, strongly absorbing resorufin (ex/em ∼570/585 nm) or Amplex UltroxRed (ex/em ∼568/581 nm), respectively
• These oxidized reaction products have a broad red emission with little interference from blue and green autofluorescence found in most biological samples
• Because H 2 O 2 is produced in many different enzymatic reactions, the Amplex Red and UltraRed reagents coupled with HRP can be used to detect the activity of many different enzymes
• These reagents are also used to detect H 2 O 2 released from biological samples, including cells and isolated mitochondria
• Features of the Amplex Red reagent: • Sensitive and fast detection of HRP activity or H 2 O 2 for solution-based assays and ELISAs • Detection of H 2 O 2 produced in many different enzymatic reactions • Measurement with either fluorescence or absorbance, permitting either fluorogenic or chromogenic detection The Amplex Red reagent has been used to develop a variety of novel fluorogenic and chromogenic assays for enzymes that produce hydrogen peroxide
• When coupled with added horseradish peroxidase, these assays that generate H 2 O 2 permit the ultrasensitive quantitation of a diverse assortment of analytes, including glucose, galactose, cholesterol, glutamic acid, xanthine (or hypoxanthine), uric acid, choline, and acetylcholine
• The Amplex UltraRed reagent offers brighter fluorescence and enhanced sensitivity on a per-mole basis in peroxidase or peroxidase-coupled enzyme assays compared to Amplex Red
• The Amplex UltraRed reagent is also less sensitive to acidic pH and exhibits improved stability in the presence of H 2 O 2 or thiols such as dithiothreitol (DTT)
• For HRP-based ELISA applications, this results in at least two-fold greater sensitivity compared to Amplex Red
• Benefits of the Amplex UltraRed reagent over Amplex Red: • Brighter signals and enhanced sensitivity for HRP activity and H 2 O 2 • Less sensitive to acidic pH • Improved stability in the presence of H 2 O 2 or thiols such as DTT The Amplex Red and Amplex UltraRed reaction products resorufin and Amplex UltroxRed have high extinction coefficients so they can be measured either fluorometrically or spectrophotometrically with absorbance at ∼560 nm, providing flexibility in detection of H 2 O 2 , various enzyme assays, and HRP-based ELISAs
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