Microplate Assays for Enzyme Activity

EnzCheck Assays provide a rapid and convenient way to measure a range of enzyme activities using fluorescent reporters in a robust assay format. Each assay kit provides the necessary assay buffers, uses a simple protocol and defines the optimum wavelength for sensitive detection. The microplate format is convenient for high throughput analysis using a 200 µL assay volume. We offer assays for proteases, collagenases, elastases, lysozyme, reverse transcriptase, and RNA-dependent RNA polymerase.

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Protease assays

The EnzChek Protease Assay Kits contain a casein derivative that is heavily labeled with either the green-fluorescent BODIPY FL or red-fluorescent BODIPY TR-X dye. The conjugates typically exhibit <3% of the fluorescence of the corresponding free dyes. Following the assay, the observed increase in fluorescence is directly proportional to protease activity. Because EnzChek assays do not involve any separation steps, they can be used for continuous measurement.

Other enzyme assays

We've also developed EnzChek Assay Kits for gelatinase/collagenase, elastase, lysozyme, and reverse transcriptase that are reported by intramolecular self-quenched substrates, allowing continuous, sensitive monitoring of enzyme kinetics.

Selection guides
 EnzChek Protease Assay Kit, Green FluorescenceEnzChek Protease Assay Kit, Red Fluorescence
TargetProteaseProtease
ReporterBODIPY FL conjugateBODIPY TR-X conjugate
Ex/Em (nm)505/513589/617
Live cellNoNo
LysateYesYes
Purified enzymeYesYes
UsageDetection limit varies by proteaseDetection limit varies by protease
ComponentsIncludes assay buffersIncludes assay buffers
Format1,000 assays using 200 µL reaction volume1,000 assays using 200 µL reaction volume
Protocol outline
  1. Lyse cells, isolate supernatant.
  2. Prepare substrate and apply to wells.
  3. Add samples to wells.
  4. Incubate samples.
  5. Measure fluorescence.
  6. Determine activity using standard curve.
  1. Lyse cells, isolate supernatant.
  2. Prepare substrate and apply to wells.
  3. Add samples to wells.
  4. Incubate samples.
  5. Measure fluorescence.
  6. Determine activity using standard curve.
Cat. No.E6638E6639
 EnzChek Gelatinase/Collagenase Assay KitEnzChek Elastase Assay KitEnzChek Lysozyme Assay KitEnzChek Reverse Transcriptase Assay Kit
TargetCollagenase/GelatinaseElastaseLysozymeReverse transcriptase
ReporterFluorescein conjugateBODIPY FL ConjugateFluorescein conjugatePicoGreen reagent
Ex/Em (nm)495/515505/515495/515480/520
Live cellNoNoNoNo
LysateYesYesYesYes
Purified enzymeYesYesYesYes
UsageLower detection limit 2 x 10–4 U/mLLower detection limit 5 x 10–3 U/mLLower detection limit 20 U/mLLower detection limit 2 x 10–3 U/mL
ComponentsIncludes assay buffers and controlsIncludes assay buffers, inhibitor, and controlsIncludes assay buffers and controlsIncludes assay buffers
Format250–1,000 assays depending on substrate concentration600 assays using 200 µL reaction volume400 assays using 100 µL reaction volume1,000 assays using 200 µL reaction volume
Protocol outline
  1. Apply DQ gelatin substrate to wells.
  2. Add enzyme-containing samples to wells.
  3. Incubate.
  4. Measure fluorescence.
  1. Apply DQ elastin substrate to wells.
  2. Add enzyme-containing samples to wells.
  3. Incubate.
  4. Measure fluorescence.
  1. Apply DQ lysozyme substrate to wells.
  2. Add enzyme-containing samples wells.
  3. Incubate.
  4. Measure fluorescence.
  1. Anneal template and primer.
  2. Add annealed material to reaction mixture, then add to wells.
  3. Add samples and standards to wells.
  4. After 1 hr, add EDTA to stop reaction.
  5. Add the PicoGreen dsDNA working solution.
  6. Incubate 5 min.
  7. Measure fluorescence.
Cat. No.E12055E12056E22013E22064
Proteases
 EnzChek Protease Assay Kit, Green FluorescenceEnzChek Protease Assay Kit, Red Fluorescence
TargetProteaseProtease
ReporterBODIPY FL conjugateBODIPY TR-X conjugate
Ex/Em (nm)505/513589/617
Live cellNoNo
LysateYesYes
Purified enzymeYesYes
UsageDetection limit varies by proteaseDetection limit varies by protease
ComponentsIncludes assay buffersIncludes assay buffers
Format1,000 assays using 200 µL reaction volume1,000 assays using 200 µL reaction volume
Protocol outline
  1. Lyse cells, isolate supernatant.
  2. Prepare substrate and apply to wells.
  3. Add samples to wells.
  4. Incubate samples.
  5. Measure fluorescence.
  6. Determine activity using standard curve.
  1. Lyse cells, isolate supernatant.
  2. Prepare substrate and apply to wells.
  3. Add samples to wells.
  4. Incubate samples.
  5. Measure fluorescence.
  6. Determine activity using standard curve.
Cat. No.E6638E6639
Other enzymes
 EnzChek Gelatinase/Collagenase Assay KitEnzChek Elastase Assay KitEnzChek Lysozyme Assay KitEnzChek Reverse Transcriptase Assay Kit
TargetCollagenase/GelatinaseElastaseLysozymeReverse transcriptase
ReporterFluorescein conjugateBODIPY FL ConjugateFluorescein conjugatePicoGreen reagent
Ex/Em (nm)495/515505/515495/515480/520
Live cellNoNoNoNo
LysateYesYesYesYes
Purified enzymeYesYesYesYes
UsageLower detection limit 2 x 10–4 U/mLLower detection limit 5 x 10–3 U/mLLower detection limit 20 U/mLLower detection limit 2 x 10–3 U/mL
ComponentsIncludes assay buffers and controlsIncludes assay buffers, inhibitor, and controlsIncludes assay buffers and controlsIncludes assay buffers
Format250–1,000 assays depending on substrate concentration600 assays using 200 µL reaction volume400 assays using 100 µL reaction volume1,000 assays using 200 µL reaction volume
Protocol outline
  1. Apply DQ gelatin substrate to wells.
  2. Add enzyme-containing samples to wells.
  3. Incubate.
  4. Measure fluorescence.
  1. Apply DQ elastin substrate to wells.
  2. Add enzyme-containing samples to wells.
  3. Incubate.
  4. Measure fluorescence.
  1. Apply DQ lysozyme substrate to wells.
  2. Add enzyme-containing samples wells.
  3. Incubate.
  4. Measure fluorescence.
  1. Anneal template and primer.
  2. Add annealed material to reaction mixture, then add to wells.
  3. Add samples and standards to wells.
  4. After 1 hr, add EDTA to stop reaction.
  5. Add the PicoGreen dsDNA working solution.
  6. Incubate 5 min.
  7. Measure fluorescence.
Cat. No.E12055E12056E22013E22064

Expertly detect fluorescence with Thermo Scientific plate readers

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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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