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 autofluorescene in typical cellular samples and the signal shows little sensitivity to pH or reducing agents.

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.

Amplex® Red Glucose⁄Glucose Oxidase Assay Kit
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 Kit Amplex® Red Glucose/Glucose Oxidase Assay Kit Amplex® Red Uric Acid/Uricase Assay Kit
Target Galactose/Galactose Oxidase Glucose/Glucose Oxidase Uric acid/Uricase
Reporter Amplex® Red reagent Amplex® Red reagent Amplex® Red reagent
Ex/Em (nm) 571/585 571/585 571/585
Live cell No No No
Lysate Yes Yes Yes
Purified enzyme Yes Yes Yes
Usage Lower detection limit 2 x 10–3 U/mL Lower detection limit 5 x 10–5 U/mL Lower detection limit 2 x 10–4 U/mL
Components Includes assay buffers and controls Includes assay buffers and controls Includes assay buffers and controls
Format 400 assays using 100 µL reaction volume 500 assays using 100 µL reaction volume 400 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. A22179 A22189 A22181
  Amplex® Acetylcholine/
Acetlycholinesterase Assay Kit
Amplex® Red Monoamine Oxidase Assay Kit Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit
Target Acetylcholinesterase Monoamine oxidase Glutamic Acid/Glutamate Oxidase
Reporter Amplex® Red reagent Amplex® Red reagent Amplex® Red reagent
Ex/Em (nm) 571/585 571/585 571/585
Live cell No No No
Lysate Yes Yes Yes
Purified enzyme Yes Yes Yes
Usage Ultrasensitive method for continuously monitoring acetylcholinesterase (AChE) activity Lower detection limit 12 x 10–6 U/mL Lower detection limit 4 x 10–5 U/mL
Components Includes assay buffers and controls Includes assay buffers and controls Includes assay buffers and controls
Format 500 assays using 200 µL reaction volume 500 assays using 100 µL reaction volume 200 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. A12217 A12214 A12221
  Amplex® Red Neuraminidase (Sialidase) Assay Kit Amplex® Red Xanthine/Xanthine Oxidase Assay Kit Amplex® Red Catalase Assay Kit Amplex® Red Sphingomyelinase Assay Kit
Target Neuraminidase (sialidase) Xanthine/Xanthine oxidase Catalase Sphingomyelinase
Reporter Amplex® Red reagent Amplex® Red reagent Amplex® Red reagent Amplex® Red reagent
Ex/Em (nm) 571/585 571/585 571/585 571/585
Live cell No No No No
Lysate Yes Yes Yes Yes
Purified enzyme Yes Yes Yes Yes
Usage Lower detection limit 2 x 10–4 U/mL Lower detection limit 1 x 10–4 U/mL Lower detection limit 5 x 10–4 U/mL Lower detection limit 8 x 10–7 U/mL
Components Includes assay buffers and controls Includes assay buffers and controls Includes assay buffers and controls Includes assay buffers and controls
Format 400 assays using 100 µL reaction volume 400 assays using 100 µL reaction volume 400 assays using 100 µL reaction volume 500 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. A22178 A22182 A22180 A12220
For Research Use Only. Not for use in diagnostic procedures.