This protocol begins by adding incubation buffer, FXI casein solution and sample containing the protease of interest to a micro centrifuge tube, and then gently mixing and incubating at 37 degrees Celsius for 60 minutes in the dark. To stop the proteolytic reaction at the end of the incubation period, add TCA solution gently mix and incubate at 37 degrees Celsius for 30 minutes again in the dark. This step will cause the unreactive substrate to precipitate.
Centrifuge the sample for 10 minutes. At 10, 000 Gs, remove the supernatant, which contains the acid soluble FXI labeled fragments. Transfer the supernatant to a vet or multiple well plate dilute with assay buffer and read the sample on a fluorimeter.
Hi, I'm Carrie Cup Vineyard and I'm a scientist at our Sigma Aldridge DeKalb facility in St.Louis, Missouri. Today I will be showing you a procedure for fluoro metric protease activity determination. We use this procedure in our quality control lab to determine low protease activity.
So let's get started For this procedure, the incubation buffer, assay buffer, and 0.6 normal trichloroacetic acid or TCA solution are provided as ready-to-use solutions. Aliquot the solution of fite labeled Cain into smaller volumes and store at minus 20 degrees Celsius. Making sure to protect from light by using amber vials or wrapping clear tubes in foil Ali.
Quoting the fite Cain substrate minimizes the need for repeated freeze thaw cycles, repeated freeze thaw cycles or vigorous shaking or mixing of the fite Cain could lead to separation of the fite from the Cain, resulting in higher background. If calibrating the fluorimeter or determining the linear range for the fit e signal, prepare the fit control solution. Make the solution fresh by reconstituting in assay buffer to the appropriate concentration.
Once ready, protect the solution from light. Finally, prepare the tripsin protease control by adding 100 microliters of one millimolar hydrochloric acid to the vial of lyophilized trypsin, and to mix briefly to ensure the trypsin is dissolved. Storing the tripsin under acidic conditions increases its stability.
Note that the Tripsin control solution is temperature sensitive and is not stable at low concentrations. Next, start preparing all of the assay samples, which include test samples and control and blank throughout the assay. Keep the samples protected from light for each test sample.
Combine in a micro centrifuge tube, 20 microliters of incubation buffer, 20 microliters of the fit c kian substrate and 10 microliters of the test sample. One may use a clear micro centrifuge as long as the sample is incubated in the dark. Note also that test samples, which have high protease activity will need to be diluted.
The tripsin control sample is used as a positive control that the assay is performing properly to determine the detection limit or to create a general standard curve to prepare the tripsin control, combine one part acidified trypsin to nine parts of incubation buffer or the buffer used for the test samples and mix well. The final working concentration of trypsin is 20 micrograms per milliliter. If using a different specific protease, prepare the control solution with that specific protease and an appropriate buffer.
Continue to prepare the control sample by combining 20 microliters of incubation buffer, 20 microliters of fz cain substrate and 10 microliters of the trypsin control in a micro centrifuge tube. It is recommended to run at least one control sample of five nanograms trypsin with each assay or to use serial dilution. The last sample is the blank, prepared by combining 20 microliters of incubation buffer, 20 microliters of the Fitz Caine substrate and 10 microliters of ultrapure water in a micro centrifuge tube.
With the three types of samples ready, gently mix each tube and incubate at 37 degrees Celsius in the dark for 60 minutes. Be careful not to mix too vigorously as excessive turbulence may cause high fluorescence background and reduce the sensitivity of the assay. One can incubate up to 24 hours to increase sensitivity, but longer incubations are not recommended as the FXI casein may begin to degrade.
Again, leading to high fluorescence background when the incubation is complete, use appropriate protective equipment to add 150 microliters of the TCA solution to each micro centrifuge tube. Upon incubation with TCA one should observe a white haze present in the tube, which is the precipitated, undigested and insoluble cain mix gently and incubate at 37 degrees Celsius in the dark for 30 minutes. At the end of the incubation with the TCA solution centrifuge the tubes for 10 minutes at 10, 000 Gs at room temperature.
The supernatant contains the acid soluble fite labeled Cain fragments used for the fluorescence measurement pipette gently from the top of the tube so that none of the precipitate in the pellet is drawn into the pipette tip, which will cause a false positive readout during the following fluorescent detection to measure the protease activity using a fluorimeter combined 10 microliters of the TCA supernatant with one milliliter of the assay buffer in a tube and mix gently. In addition, dilute the FE standard for analysis by adding 10 microliters of FE to one milliliter of assay buffer. The FE control and Tripsin standard give a fluorescence level that the sample should not exceed the solution of the TC.A supernatant and assay buffer may be stored in the dark at two to eight degrees Celsius for up to 24 hours before measuring the fluorescence.
If measuring using a vete transfer one milliliter of the solution to a suitable vete, if a large number of samples are to be run a 96 well plate and fluorescent plate reader may be used to acquire fluoro metric data. In this assay, fluorescein isothiocyanate is provided as a control for possible instrument calibration. Shown here is a representative linearity range of the FS e signal following the described procedure.
Using this kit with an incubation time of 30 minutes for the first incubation step yields this typical standard curve for trypsin and control samples ranging from 1.5 nanograms to 25 nanograms. Fluorescence measurements were made in a multi-well plate. The limit of detection of the assay is the amount of protease that produces a significant fluorescence reading above the value obtained with the blank sample.
Today we've shown you how to use the Sigma Aldridge protease determination kit to detect Fluor metric protease activity in a biological sample. While this kit is optimized for your use, it's important to remember that for your particular proteases, you may need slight modifications. So that's it.
Thanks for watching and good luck with your experiments.
