The Thermo Scientific Pierce Quantitative Protease Assay Kits measure total protease activity in samples, providing a means to assess the progress of protease isolation procedures or quantify protease contamination in biological samples.
These Colorimetric and Fluorescent Protease Assay Kits (formerly "QuantiCleave") 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
- 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
Features of the Colorimetric Kit (Part No. 23263):
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)
Colorimetric protease assay response curves. The Thermo Scientific Pierce Colorimetric Protease Assay Kit (Part No. 23263) was used to measure the activity of V-8 protease and submaxillary protease for digestion of casein substrate by comparison to the supplied trypsin standard.
Features of the Fluorescent Kit (Part No. 23266):
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)
Fluorescent protease assay standard curves in FRET and FP modes. The Thermo Scientific Pierce Fluorescent Protease Assay Kit (Part No. 23266) measures relative protease activity for digestion of FITC-conjugated casein substrate by comparison to a supplied trypsin standard. The assay can be measure in either FRET or FP modes.