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Thermo Scientific™ Pierce™ Colorimetric Protease Assay Kit

Determine total protease activity in biological samples via fluorescent or colorimetric detection by measuring digestion of labeled casein substrate.

Manufacturer:  Thermo Scientific™ 23263

Catalog No. PI23263

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Thermo Scientific™ Pierce Protease Assay Kits measure total protease activity in samples, providing a means of assessing the progress of protease isolation procedures or quantifying protease contamination in biological samples.

These Colorimetric and Fluorescent Protease Assay Kits comprise two different methods for measuring total protease activity in biological samples. Both kits can be used to assess the progress of protease purification procedures or to quantify protease contamination in protein samples. The kits use succinylated or fluorescein-tagged casein as a protease substrate. Proteolysis of the substrate results in peptides whose amino groups or altered fluorescence properties enable detection in the respective kits. Protease activity is quantified by comparison to trypsin standard included in each kit.


  • Casein substrate – measure activity of any protease that cleaves casein into peptide fragments

  • Trypsin standard – quantify protease activity relative to trypsin, a universally accepted reference

  • Sensitive – 1000 times more sensitive than assays that use unmodified forms of casein

  • Simple and fast – test tube and microplate protocols are completed in less than one hour

  • Homogenous – addition steps only; no separation, transfer or stop steps required

  • Customizable – easily adapt time, temperature and pH to optimize sensitivity for proteases of interest

The Colorimetric Protease Assay Kit (Part No. 23263) uses fully succinylated casein as a substrate for the assay. Hydrolysis of this substrate in the presence of protease results in the release of peptide fragments with free amino-terminal groups. These peptides are reacted with trinitrobenzene sulfonic acid (TNBSA), followed by measurement of the absorbance increase that results from the formation of yellow colored TNB-peptide adducts.
  • Substrate: succinylated casein (not available separately)
  • Basis of assay: detection of primary amines resulting from proteolysis
  • Detection reagent: trinitrobenzene sulfonic acid (TNBSA)
  • Measurement: absorbance at 450nm (plate reader or spectrophotometer)
The Fluorescent Protease Assay Kit (Part No. 23266) includes fluorescein-labeled casein as a substrate for assessing protease activity in a sample by either fluorescence resonance energy transfer (FRET) with a standard fluorometer or fluorescence polarization (FP) with capable instrumentation. FTC-Casein is native casein that has been labeled using a large molar excess of fluorescein isothiocyanate (FITC). Fluorescence properties of this heavily-labeled, intact protein substrate change dramatically upon digestion by proteases, resulting in a measurable indication of proteolysis.
  • Substrate: fluorescein-labeled casein (FITC-casein); available separately as Part No. 23267
  • Basis of assay: change in fluorescence resonance (FRET) or fluorescence polarization (FP)
  • Measurement: instrument with fluorescein excitation and emission filters (485/538nm)


Modified casein, trypsin standard and Tris-buffered saline (both kits); TNBSA solution (Part No. 23263 only)

Recommended for:

Assess functional integrity and overall progress of protease purification; Quantify protease contamination in protein samples; Characterize the activity of unusual proteases compared to trypsin; Evaluate buffer conditions for their affects on protease function and activity



96-well plate
Sufficient For: 250 microplate assays using a standard plate reader (absorbance at 450 nm)
• Succinylated Casein, 5 x 10 mg
• TNBSA Solution, 2 mL
• TPCK Trypsin Standard, 50 mg
• Borate Buffer Pack (makes 500 mL), 1 pack

Store at 4°C.
Protein Activity Assay
Protease Substrate
250 Test Kit
Protein-Based Substrate
Microplate Reader
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