Thermo Scientific™ Pierce™ Fluorescent Protease Assay Kit

Description

• 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
• Highlights: 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) Includes: 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