Protease Detection Kit, Colorimetric, BioAssay™

Kits and Assays / Kits-Molecular Biology, BioAssay™

4°C

The BioAssay™ Protease Detection Kit is a colorimetric assay used to detect protease activity. The proprietary substrate supplied in the kit is susceptible to a wide range of proteases, e.g., collagenase, Proteinase K, papain, pepsin, bromelain, ficin, trypsin and chymotrypsin. The substrate is a cross-linked matrix containing protein and a dye-protein conjugate. The matrix is supplied in ready to use vials.

Test samples containing putative protease activity are aliquoted into the vials and incubated at 37°C. Protease activity is detected spectrophotometrically with increasing optical density proportional to increasing enzyme activity.

Advantages:
• Simple to use. Just add sample, incubate and read absorbance.
• No Precipitation Required
• No Centrifugation Required
• No Hazardous Materials Required
• Detection Limits as low as 1ng
• Fast...Protease Detection and Quantitation in minutes

Specificity:
Serine, metallo, aspartate and cysteine proteases

Introduction:
Interest in proteases has increased with the realization that they play key roles in rheumatoid and cancer metastasis. Tumor progression depends on remodeling of basement membrane prior to invasion and angiogenesis. Some inhibitors of the proteases which catalyze these processes have shown good anti-tumor activity without the side effects of cytotoxic drugs. More recently, protease inhibitors have proved effective as anti-viral agents most notably in the treatment of AIDS patients.

In addition to those investigators who target proteases for medical purposes, biochemists are generally concerned with protection of their valuable proteins from unwanted degradation by contaminating proteases. These workers need to verify the presence or absence of proteases in their preparations. During the manufacture of proteases for industrial use, aerosolized enzyme has become a health concern. Consequently, there is a need for low-cost, simple to use, rapid test systems for monitoring environmental contamination.

Modern methods have supplanted the classical three-step assay-digestion, TCA precipitation, and detection of TCA soluble peptides by UV absorbance, the Folin-Ciocalteu reagent, or other means. Two currently popular assays are based on derivitized casein. In 1984, Twining introduced a widely applicable and sensitive assay based on fluorescein isocyanate-labeled casein. Another assay employs azocoll, an insoluble protein-dye conjugate, which when hydrolyzed yields soluble, colored peptides. The non-hydrolyzed insoluble material must be removed by filtration or centrifugation. These assay procedures are thus labor intensive and cumbersome for high volume assay procedures.

In 1992, Hatakeyama, et al., reported an assay based on succinyl-casein and the TNBS reagent for detection of liberated amino groups. This assay can be run with the multi-well plate reader. Disadvantages include failure to detect collagenases, background due to reactive amines or thiols, and interference from particulates in the sample.

The BioAssay™ Protease Detection Kit utilizes a mixture of gelatin and albumin cross-linked in the presence of sulfaniloazo-albumin. The translucent solid is mechanically stable even at elevated temperature. Twenty-four hour backgrounds without protease are acceptably low, when the assay mixture contains a biocide. A broad range of proteases including collagenase, papain, bromelain, trypsin, chymotrypsin, proteinase K, and pronase, exhibit good activity against this protein matrix.

Assay Method:
In a typical assay, 500ul of reaction buffer and 100ul of enzyme solution are added to the supplied vials (a mixture of gelatin and albumin cross-linked in the presence of sulfaniloazo-albumin) and incubated at 37°C. To stop the reaction, 500ul of 0.2N NaOH is added to each vial and then the absorbance at 450nm of the aqueous phase is measured.

Kit Components:
P9055-01: Premade matrix 48x200ul
P9055-02: Trypsin Control 1x500ul 1x500ul Concentration: 0.7mg/ml (1420 BAEE units/mg) Supplied as a liquid in reaction buffer (10mM Tris-HCl, pH 8.0).

Materials Required But Not Provided:
0.2N NaOH
Spectrophotometer capable of reading 450nm

Storage and Stability:
May be stored at 4°C. Do not freeze. Stable for 3 months after receipt.