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
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)
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