PDQ Protease Assay | 0201

The PDQ (Protease Determine Quick) Protease Assay is a colorimetric assay used to locate protease pastime in aqueous samples. The proprietary substrate responds to a extensive variety of proteases consisting of serine, metallo, aspartate and cysteine proteases consisting of collagenase, proteinase K, papain, pepsin, bromelin, ficin, trypsin and chymotrypsin. PDQ™ may be used with only some easy steps to degree protease pastime and calls for no centrifugation. The substrate is a cross-related matrix containing protein substrate and a dye-protein conjugate. Protease pastime is detected spectrophotometrically with growing optical density proportional to growing enzyme pastime and might locate nanogram quantities.

Instructions for Use

•  Allow the vials to return back to room temperature earlier than strolling the assay. Pour off garage answer.
• To assemble a preferred curve, put together a ten-fold dilution collection of the manage protease. This manage answer ought to comprise the protease for which the take a look at is being run. Trypsin is furnished as a trendy protease manage. The trypsin may be utilized in a ten-fold dilution collection; e.g., four hundred to 0.four BAEE units/ml (280 – 0.28 µg/ml). Any buffer may be used with the PDQ matrix. . The preferred trypsin response buffer is 10mM Tris-Cl, pH 8.0.
•  Add 0.five ml of every manage answer to replicate vials.
•  Prepare take a look at samples containing putative protease pastime withinside the equal buffer as the manage protease. Add 0.five ml of the take a look at pattern to replicate vials.
•  Replace snap caps onto vials.
• Incubate vials at 37ºC for 1 h. (A shorter or longer incubation time can also additionally be required.
•  Add 0.five ml 0.2N NaOH to every vial to forestall the response and increase color. Either lightly swirl the vial or update the cap and invert the vial as soon as to combine contents.
•  Transfer the contents (being cautious now no longer to disturb the semi-stable matrix) to a preferred 1 ml cuvette. (Transfer 0.2 ml of liquid if the use of a microplate reader.)
•  Record the absorbance at 450 nm.
• Average the consequences from reproduction vials and use the manage samples to generate a preferred curve, plotting concentration (or quantity) of manage protease vs. A450.

Compare take a look at pattern consequences

towards the usual curve to achieve the quantity of protease pastime.  Buffers that may be used to put together take a look at samples can also additionally variety in pH from four to 10.  The quantity of time required for incubation can be numerous to optimize the assay. Up to 24 hr incubations are feasible as a way to boom the detection limits of the assay. Alternatively, for packages with excessive quantities of enzyme, 15 min can also additionally suffice. The genuine situations ought to be decided empirically. As with any assay, it’s miles essential to run a preferred curve with manage protease on every occasion the assay is accomplished with unknown protease samples. When the use of the vials, the stopped response contents have to be transferred to a cuvette to degree the absorbance. The switch of the contents farfar from the PDQ matrix ought to be completed as quickly as feasible after the addition of NaOH, for the reason that NaOH will degrade the matrix over time.

However, the contents may be left at the matrix for up to at least one hour earlier than switch with out compromising the accuracy of the take a look at. Once the stopped response contents are transferred farfar from matrix, they may be left for as much as 24 hrs covered (to preserve out light) at room temperature earlier than measuring the absorbance with out lack of sign. To use the Colorimetric Assay for opaque samples, run the response as described. Remove the response liquid after which lower back digest the last substrate matrix with Trypsin, Subtilisin or Proteanase K. The sign generated from the lower back digestion is inversely proportional to the quantity of enzyme pastime. I