Datasheets

Product Description

The PDQ? (Protease Determine Quick) Protease Assay is a colorimetric assay used to detect protease activity in aqueous samples. The proprietary substrate responds to a wide range of proteases including serine, metallo, aspartate and cysteine proteases such as collagenase, proteinase K, papain, pepsin, bromelin, ficin, trypsin and chymotrypsin. PDQ? can be used with just a few simple steps to measure protease activity and requires no centrifugation. The substrate is a cross-linked matrix containing protein substrate and a dye-protein conjugate. Protease activity is detected spectrophotometrically with increasing optical density proportional to increasing enzyme activity and can detect nanogram quantities.

The PDQ? Fluorescent Protease Assay employs the same protease lattice matrix as the Colorimetric PDQ? Protease Assay. Fluorescein Isothiocyanate (FITC) is incorporated into the substrate as an indicator of enzymatic digestion of the substrate, allowing for detection of sub-nanogram quantities of protease activity in aqueous samples. The fluorescent-based substrate responds to the same broad spectrum of proteases as the Colorimetric PDQ? Assay. Protease activity is detected fluorophotometrically with increasing fluorescence proportional to increasing enzyme activity.

Each kit is supplied with a trypsin solution for generating standard curves (BAEE equivalent units). PDQ? is supplied in 48 ready-to-use plastic vials.

Instructions for Use

1.         Allow the vials to come to room temperature before running the assay. Pour off storage solution.

2.         To construct a standard curve, prepare a ten-fold dilution series of the control protease. This control solution should contain the protease for which the test is being run (e.g., use proteinase K in the control dilution series if this is the protease being tested for). Trypsin is supplied as a general protease control. The trypsin can be used in a ten-fold dilution series; e.g., 400 ?> 0.4 BAEE units/ml (280 - 0.28 g/ml). Any buff er can be used with the PDQ? matrix. (See Technical Considerations). The standard trypsin reaction buff er is 10mM Tris-Cl, pH 8.0.

3.         Add 0.5 ml of each control solution to duplicate vials.

4.         Prepare test samples containing putative protease activity in the same buffer as the control protease. Add 0.5 ml of the test sample to duplicate vials.

5.         Replace snap caps onto vials.

6.         Incubate vials at 37sC for 1 hr. (A shorter or longer incubation time may be required. See Technical Considerations).

7.         Add 0.5 ml 0.2N NaOH to each vial to stop the reaction and amplify color. Either gently swirl or tap the vial or replace the cap and invert the vial once to mix contents. (Add 1.5ml 0.1N NaOH if assaying the contents in standard cuvettes).

8.         Transfer the contents (being careful not to disturb the semi-solid matrix) to semi-micro or standard cuvettes.

9.         For spectrophotometric assays, record absorbance at 450 nm. For fluorimetric assays, record the fluorescence for FITC settings (485 nm excitation/ 535 nm emission).

10.       Average the results from duplicate vials and use the control samples to generate a standard curve, plotting concentration (or amount) of control protease vs. A450 or fluorescence. Compare test sample results against the standard curve produced by the control protease dilution series to obtain semi-quantitative protease activity data.

Technical Considerations

1.         Buffers that can be used to prepare test samples may range in pH from 4 to 10.

2.         The amount of time required for incubation can be varied to optimize the assay. Up to 24 hr incubations are possible which will increase the detection limits of the assay. Alternatively, for applications with high amounts of enzyme, 15 min may suffice. The exact conditions should be determined empirically.

3.         As with any assay, it is necessary to run a standard curve with control protease every time the assay is performed with ?unknown? protease samples.

4.         When using the vials, the stopped reaction contents must be transferred to a cuvette to measure the absorbance. The transfer of the contents away from the PDQ? matrix should be done as soon as possible after the addition of NaOH, since the NaOH will degrade the matrix over time. However, the contents can be left on the matrix for up to one hour before transfer without compromising the accuracy of the test.

5.         Once the stopped reaction contents are transferred away from matrix, they can be left for up to 24 hrs covered (to keep out light) at room temperature before measuring the absorbance without loss of signal.