Protease Fluorescent Detection Kit, PF0100
 Protease Colorimetric Detection Kit, PC0100

Now there are two easy-to-use kits to detect trace amounts of protease or determine total protease activity using the same methodologies utilized by Sigma's QC department for years. Sigma has used these protocols to test thousands of samples for proteolytic activity.

The Protease Detection Kits are complete kits for detection of primary or trace protease activity by your choice of fluorometric or colorimetric detection. Everything you need, including control, standards, buffers, and substrate, is included. For convenience the assay can be performed in either a cuvette or microplate format. Each kit contains enough reagents for 200 x 1 mL assays. For greater sensitivity when measuring low levels of protease activity, the Protease Fluorescent Detection kit (PF0100) is recommended. For more accurate quantification of primary protease activity, the Protease Colorimetric Detection kit is (PC0100) recommended.

Both the Fluorometric Detection Kit and the Colorimetric Detection Kit have been tested against representative samples of all four types of protease classes (serine, aspartic, cysteine and metalloproteases) to ensure broad application suitability.

The Protease Fluorescent Detection Kit is based on an assay method using highly labeled fluorescein isothiocyanate (FITC) casein as the substrate. This is a modified version of the procedure for protease detection as described by Twining.[1] The kit will detect 0.5 µg/mL of protease (approx. 5 ng trypsin) and has been tested for detection of all four proteolytic classes (serine, aspartic, cysteine and metalloproteases). The sensitivity level can be increased by using longer incubation times of up to 24 hours.

Detection limits for the Protease Fluorescent Detection Kit

Protease	Protease Class	Detection Limits	Cleavage Site(s)1
Trypsin	Serine	< 1.0 ng	C-terminal sites of Arg and Lys
Pepsin	Aspartic	1.5 ng	Hydrophobic, preferably aromatic, residues in P1 and P1' positions.
Papain	Cystine	1.5 ng	Hydrolysis of proteins with broad specificity for peptide bonds, but preference for an amino acid bearing a large hydrophobic side chain at the P2 position. Does not accept Val in P1'
Carboxypeptidase A	Metalloproteinase	5.0 ng	Release of a C-terminal amino acid, but little or no action with -Asp, -Glu, -Arg, -Lys or -Pro

Sensitivity of the Protease Fluorescent Detection Kit (PF0100) using Trypsin (T6567) Each of the trypsin samples was incubated with the proprietary formulation of the FITC-casein substrate for 17 hours for increased sensitivity at 37°C and measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a microtiter plate.		Typical activity curve using trypsin Each of the trypsin samples was incubated with the proprietary formulation of the FITC-casein substrate for 30 minutes at 37°C and measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a microtiter plate.

• Incubation Buffer
• Assay Buffer
• FITC-casein substrate
• 0.6 N TCA solution
• Trypsin, protease control
• Fluorescein isothiocyanate (FITC) control

Product #	Product Name	Package Size	Technical Manual
PF0100	Protease Fluorescent Detection Kit	1 KT

Reference

1. Twining, S.S., Fluorescein Isothiocyanate-Labeled Casein Assay for Proteolytic Enzymes. 1984. Anal. Biochem., 143, 30-34 (1984).

The Protease Colorimetric Detection Kit provides robust, linear response with convenient colorimetric detection.

The kit is based on the cleavage of casein into trichloroacetic acid-soluble peptides containing tyrosine and tryptophan residues. These peptides react with Folin-Ciocalteu reagent to give a characteristic blue color that can be measured at 660 nm. The kit will detect approximately 0.1 units/ml of protease (approx. 2 µg), and has been tested for detection of all four proteolytic classes (serine, aspartic, cysteine and metalloproteases).

• Protease Detection Substrate: Casein
• Folin & Ciocalteu’s phenol reagent (Product Code F9252)
• 500 mM Sodium carbonate solution
• Enzyme diluent
• 0.6 N TCA
• Tyrosine standard solution
• Protease from Bacillus polymyxa control

Product #	Product Name	Package Size	Technical Manual
PC0100	Protease Colorimetric Detection Kit	1 KT

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