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, and reverse transcriptase.

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.

EnzChek® Elastase Assay Kit
Assay of porcine pancreatic elastase using the EnzChek® Elastase Assay KitDye-conjugated elastin (25 μg/mL) from the kit was incubated with various concentrations of elastase for the specified incubation times, and fluorescence was measured.

Selection guides
  EnzChek® Protease Assay Kit, Green Fluorescence EnzChek® Protease Assay Kit, Red Fluorescence
Target Protease Protease
Reporter BODIPY® FL conjugate BODIPY® TR-X conjugate
Ex/Em (nm) 505/513 589/617
Live cell No No
Lysate Yes Yes
Purified enzyme Yes Yes
Usage Detection limit varies by protease Detection limit varies by protease
Components Includes assay buffers Includes assay buffers
Format 1,000 assays using 200 µL reaction volume 1,000 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. E6638 E6639
  EnzChek® Gelatinase/Collagenase Assay Kit EnzChek® Elastase Assay Kit EnzChek® Lysozyme Assay Kit EnzChek® Reverse Transcriptase Assay Kit
Target Collagenase/Gelatinase Elastase Lysozyme Reverse transcriptase
Reporter Fluorescein conjugate BODIPY®FL Conjugate Fluorescein conjugate PicoGreen® reagent
Ex/Em (nm) 495/515 505/515 495/515 480/520
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 5 x 10–3 U/mL Lower detection limit 20 U/mL Lower detection limit 2 x 10–3 U/mL
Components Includes assay buffers and controls Includes assay buffers, inhibitor, and controls Includes assay buffers and controls Includes assay buffers
Format 250–1,000 assays depending on substrate concentration 600 assays using 200 µL reaction volume 400 assays using 100 µL reaction volume 1,000 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. E12055 E12056 E22013 E22064
For Research Use Only. Not for use in diagnostic procedures.