Microplate Assays Using Metabolites and Analytes

Amplex Red Assays

Amplex Red reagent is a colorless substrate that provides exquisite sensitivity in enzyme-coupled reactions to detect a diverse assortment of analytes, including glucose, galactose, cholesterol, glutamic acid, xanthine (or hypoxanthine), uric acid, choline and acetylcholine, as well as hydrogen peroxide.

Assays using Amplex Red as the substrate result in an increase in fluorescence which is easy to detect in a microplate format. Because the emission is red, there is little interference from autofluorescence in typical cellular samples and the signal shows little sensitivity to pH or reducing agents.

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Metabolic assays

We've developed Amplex Red reagent–based assays for the detection of key metabolites including glucose/glucose oxidase, galactose/galactose oxidase, and uric acid/uricase assays.

Neurobiology assays

Separate Amplex Red assay kits are available for continuously monitoring AChE activity or for detecting ACh; for continuous measurement of amine oxidase activity in tissue homogenates or purified preparations; and for continuously detecting glutamic acid or for monitoring glutamate oxidase activity.

Inflammation assays

Assays reported using the sensitive Amplex Red reagent have been developed for the continuous assay of neuraminidase-catalyzed hydrolysis of fetuin, for detecting xanthine or hypoxanthine (or for monitoring xanthine oxidase activity), for measuring catalase activity, and for measuring sphingomyelinase activity in solution.

glucose-oxidase-s000925
Dose–response curve and sensitivity at low levels (inset) of the Amplex Red Glucose⁄Glucose Oxidase Assay Kit.
Selection guides
 Amplex Red Galactose/Galactose Oxidase Assay KitAmplex Red Glucose/Glucose Oxidase Assay KitAmplex Red Uric Acid/Uricase Assay Kit
TargetGalactose/Galactose OxidaseGlucose/Glucose OxidaseUric acid/Uricase
ReporterAmplex Red reagentAmplex Red reagentAmplex Red reagent
Ex/Em (nm)571/585571/585571/585
Live cellNoNoNo
LysateYesYesYes
Purified enzymeYesYesYes
UsageLower detection limit 2 x 10–3 U/mLLower detection limit 5 x 10–5 U/mLLower detection limit 2 x 10–4 U/mL
ComponentsIncludes assay buffers and controlsIncludes assay buffers and controlsIncludes assay buffers and controls
Format400 assays using 100 µL reaction volume500 assays using 100 µL reaction volume400 assays using 100 µL reaction volume
Protocol outline
  1. Prepare the standard curve samples and load into microplate wells.
  2. Dilute the experimental samples into reaction buffer and load into wells.
  3. Prepare the Amplex Red working solution.
  4. Begin the reaction by adding Amplex Red solution to each assay well.
  5. Incubate.
  6. Measure the resulting fluorescence.
Cat. No.A22179A22189A22181
 Amplex Acetylcholine/
Acetylcholinesterase Assay Kit		Amplex Red Monoamine Oxidase Assay KitAmplex Red Glutamic Acid/Glutamate Oxidase Assay Kit
TargetAcetylcholinesteraseMonoamine oxidaseGlutamic Acid/Glutamate Oxidase
ReporterAmplex Red reagentAmplex Red reagentAmplex Red reagent
Ex/Em (nm)571/585571/585571/585
Live cellNoNoNo
LysateYesYesYes
Purified enzymeYesYesYes
UsageUltrasensitive method for continuously monitoring acetylcholinesterase (AChE) activityLower detection limit 12 x 10–6 U/mLLower detection limit 4 x 10–5 U/mL
ComponentsIncludes assay buffers and controlsIncludes assay buffers and controlsIncludes assay buffers and controls
Format500 assays using 200 µL reaction volume500 assays using 100 µL reaction volume200 assays using 100 µL reaction volume
Protocol outline
  1. Prepare standard curve samples and load into wells.
  2. Dilute experimental samples into reaction buffer and load into wells.
  3. Prepare the Amplex Red working solution.
  4. Add Amplex Red solution to assay wells.
  5. Incubate.
  6. Measure fluorescence.
Cat. No.A12217A12214A12221
 Amplex Red Neuraminidase (Sialidase) Assay KitAmplex Red Xanthine/Xanthine Oxidase Assay KitAmplex Red Catalase Assay KitAmplex Red Sphingomyelinase Assay Kit
TargetNeuraminidase (sialidase)Xanthine/Xanthine oxidaseCatalaseSphingomyelinase
ReporterAmplex Red reagentAmplex Red reagentAmplex Red reagentAmplex Red reagent
Ex/Em (nm)571/585571/585571/585571/585
Live cellNoNoNoNo
LysateYesYesYesYes
Purified enzymeYesYesYesYes
UsageLower detection limit 2 x 10–4 U/mLLower detection limit 1 x 10–4 U/mLLower detection limit 5 x 10–4 U/mLLower detection limit 8 x 10–7 U/mL
ComponentsIncludes assay buffers and controlsIncludes assay buffers and controlsIncludes assay buffers and controlsIncludes assay buffers and controls
Format400 assays using 100 µL reaction volume400 assays using 100 µL reaction volume400 assays using 100 µL reaction volume500 assays using 200 µL reaction volume
Protocol outline
  1. Prepare standard curve samples and load into wells.
  2. Dilute experimental samples into reaction buffer and load into wells.
  3. Prepare Amplex Red working solution.
  4. Add Amplex Red solution to wells.
  5. Incubate.
  6. Measure fluorescence.
Cat. No.A22178A22182A22180A12220
Metabolic assays
 Amplex Red Galactose/Galactose Oxidase Assay KitAmplex Red Glucose/Glucose Oxidase Assay KitAmplex Red Uric Acid/Uricase Assay Kit
TargetGalactose/Galactose OxidaseGlucose/Glucose OxidaseUric acid/Uricase
ReporterAmplex Red reagentAmplex Red reagentAmplex Red reagent
Ex/Em (nm)571/585571/585571/585
Live cellNoNoNo
LysateYesYesYes
Purified enzymeYesYesYes
UsageLower detection limit 2 x 10–3 U/mLLower detection limit 5 x 10–5 U/mLLower detection limit 2 x 10–4 U/mL
ComponentsIncludes assay buffers and controlsIncludes assay buffers and controlsIncludes assay buffers and controls
Format400 assays using 100 µL reaction volume500 assays using 100 µL reaction volume400 assays using 100 µL reaction volume
Protocol outline
  1. Prepare the standard curve samples and load into microplate wells.
  2. Dilute the experimental samples into reaction buffer and load into wells.
  3. Prepare the Amplex Red working solution.
  4. Begin the reaction by adding Amplex Red solution to each assay well.
  5. Incubate.
  6. Measure the resulting fluorescence.
Cat. No.A22179A22189A22181
Neurobiology assays
 Amplex Acetylcholine/
Acetylcholinesterase Assay Kit		Amplex Red Monoamine Oxidase Assay KitAmplex Red Glutamic Acid/Glutamate Oxidase Assay Kit
TargetAcetylcholinesteraseMonoamine oxidaseGlutamic Acid/Glutamate Oxidase
ReporterAmplex Red reagentAmplex Red reagentAmplex Red reagent
Ex/Em (nm)571/585571/585571/585
Live cellNoNoNo
LysateYesYesYes
Purified enzymeYesYesYes
UsageUltrasensitive method for continuously monitoring acetylcholinesterase (AChE) activityLower detection limit 12 x 10–6 U/mLLower detection limit 4 x 10–5 U/mL
ComponentsIncludes assay buffers and controlsIncludes assay buffers and controlsIncludes assay buffers and controls
Format500 assays using 200 µL reaction volume500 assays using 100 µL reaction volume200 assays using 100 µL reaction volume
Protocol outline
  1. Prepare standard curve samples and load into wells.
  2. Dilute experimental samples into reaction buffer and load into wells.
  3. Prepare the Amplex Red working solution.
  4. Add Amplex Red solution to assay wells.
  5. Incubate.
  6. Measure fluorescence.
Cat. No.A12217A12214A12221
Inflammation assays
 Amplex Red Neuraminidase (Sialidase) Assay KitAmplex Red Xanthine/Xanthine Oxidase Assay KitAmplex Red Catalase Assay KitAmplex Red Sphingomyelinase Assay Kit
TargetNeuraminidase (sialidase)Xanthine/Xanthine oxidaseCatalaseSphingomyelinase
ReporterAmplex Red reagentAmplex Red reagentAmplex Red reagentAmplex Red reagent
Ex/Em (nm)571/585571/585571/585571/585
Live cellNoNoNoNo
LysateYesYesYesYes
Purified enzymeYesYesYesYes
UsageLower detection limit 2 x 10–4 U/mLLower detection limit 1 x 10–4 U/mLLower detection limit 5 x 10–4 U/mLLower detection limit 8 x 10–7 U/mL
ComponentsIncludes assay buffers and controlsIncludes assay buffers and controlsIncludes assay buffers and controlsIncludes assay buffers and controls
Format400 assays using 100 µL reaction volume400 assays using 100 µL reaction volume400 assays using 100 µL reaction volume500 assays using 200 µL reaction volume
Protocol outline
  1. Prepare standard curve samples and load into wells.
  2. Dilute experimental samples into reaction buffer and load into wells.
  3. Prepare Amplex Red working solution.
  4. Add Amplex Red solution to wells.
  5. Incubate.
  6. Measure fluorescence.
Cat. No.A22178A22182A22180A12220

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High-sensitivity fluorescence detection for 6-1,536 samples can be quickly performed on the Varioskan ALF or Varioskan LUX Multimode Microplate Reader using Invitrogen reagents to enable optimal detection. Take advantage of automatic dynamic range selection to get optimal gain settings for each individual well and automation capabilities for even higher throughput.

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