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A TD-700 Laboratory
Fluorometer Method for Introduction The Turner BioSystems TD-700 Laboratory Fluorometer in combination with Molecular Probes' EnzChek™ Protease Assay Kit provides fast, simple and direct fluorescence-based assays for detecting metallo-, serine, acid and sulfhydryl proteases. Detecting low levels of protease activity is important in biochemical quality-control testing as well as for basic research. Several fluorescence-based methods have been developed for detecting protease activity including the fluorescein thiocarbamoyl (FTC)-casein protease assay, in which unhydrolyzed protein must be precipitated with trichloroacetic acid, separated by centrifugation, transferred for measurement and then pH-adjusted to optimize the fluorescence signal.(1) Other methods utilize self-quenching of fluorescein dyes coupled to casein or BSA (2,3) fluorescence polarization measurements (4) or ethidium bromide binding to DNA after protease digestion of histones.(5) Molecular Probes' EnzChek Protease Assay Kits use methods that do not involve separation steps and are up to 100 times more sensitive than the FTC-casein assay.(1) The kits contain casein conjugates that are labeled with multiple BODIPY® dyes (which, unlike fluorescein, are pH-insensitive), resulting in almost total fluorescence quenching. Protease-catalyzed hydrolysis releases highly fluorescent BODIPY-labeled peptides (Figure 1). The accompanying increase in fluorescence is proportional to protease activity and can be conveniently measured in the TD-700 fluorometer equipped with a fluorescein filter kit. In addition to its utility for detecting protease contamination of culture media and other experimental samples, the assay can be used to continuously measure the kinetics of a variety of exo- and endopeptidases over a wide pH range or to measure the total substrate turnover at a fixed time following addition of the enzyme.
Materials Required
Experimental Protocol 3.1 Reagent Preparation 3.1.1 Prepare a 1.0 mg/mL substrate stock solution by adding 0.2 mL of deionized distilled H2O (ddH2O) directly to one 200 µg vial of BODIPY FL casein. This stock solution provides enough substrate for 20 protease assays. 3.1.2 Prepare 1X Digestion Buffer. Dilute 2.5 mL of the 20X Digestion Buffer with ddH2O to a final volume of 50 mL. This is enough working 1X Digestion Buffer for 20 assays [Note A]. 3.1.3 Prepare a 10 µg/mL working solution of the BODIPY FL casein. Add 0.2 mL of the stock solution prepared in step 3.1.1 to 19.8 mL of the 1X Digestion Buffer prepared in step 3.1.2. 3.2 Protease Activity Standard Curve 3.2.1 For quantitative activity determinations of purified protease preparations, a protease activity standard curve is required. If possible, use an appropriate enzyme standard of known specific activity that closely matches the protease activity being determined. A standard curve may not be relevant for samples containing one or more unknown proteases. In this case, protease activity may be expressed as fluorescence change per unit sample. For qualitative determination of the presence or absence of protease activity or contamination, a standard curve will not usually be necessary; in which case proceed to step 3.3. 3.2.2 Determine the range of enzyme response. Prepare at least four samples each containing different enzyme concentrations in 1.0 mL of the 1X Digestion Buffer (prepared in step 3.1.2) and a 1.0 mL buffer-only control sample. To each sample, add 1.0 mL of the BODIPY FL casein working solution prepared in step 3.1.3. 3.2.3 Incubate the samples for one hour, protected from light [Note B]. 3.2.4 Transfer samples to standard acrylic fluorescence cuvettes and measure the fluorescence using the TD-700 fluorometer with the fluorescein filter set (P/N 10-086R). Insert the most fluorescent sample first and calibrate the instrument sensitivity as directed in the TD-700 manual (press #2, calibrate). This procedure automatically optimizes the instrument sensitivity to match the fluorescence of the sample. 3.2.5 Measure the fluorescence of the remaining samples. To equalize any photobleaching effects, insert samples into the fluorometer for approximately equal time periods. The fluorescence value of the reagent blank (substrate + buffer only) may be subtracted from that of each sample. Corrected or uncorrected data may be used to generate a standard curve of fluorescence versus protein concentration (for example, see Figure 2). The form of the standard curve will vary with enzyme type.
3.3 Experimental Sample Analysis 3.3.1 To detect enzyme activity in a sample, dilute the experimental sample to 1.0 mL in 1X Digestion Buffer (prepared in step 3.1.2). Add 1.0 mL of the BODIPY FL casein working solution prepared in step 3.1.3. 3.3.2 Incubate the sample for one hour, protected from light. If a longer incubation time is used [Note B], the sample incubation time must be the same as that used for the standard samples in step 3.2.3. 3.3.3 Transfer the sample to a standard acrylic fluorescence cuvette and measure the fluorescence using the same instrument parameters as used in generating the standard curve (section 3.2.5). To equalize any photobleaching effects, insert samples into the fluorometer for similar time periods to those used for the standard curve measurements. 3.3.4 If the standard curve was plotted using blank-subtracted data (section 3.2.5), the reagent blank (substrate + buffer only) fluorescence value must also be subtracted from that of each of the samples. Unless it is negligible relative to experimental readings (step 3.3.3), the fluorescence reading for a substrate-free control (protease + buffer only) should be subtracted from readings for all protease-containing experimental samples to obtain the true fluorescence increase due to protease activity. Determine the protein concentration of the sample from the standard curve generated in section 3.2.5. 3.4 Protease Detection Limits Table 1 shows the approximate detection limits for a variety of proteolytic enzymes when assayed at 22° C using the EnzChek protease assay with a fluorescence microplate reader [Note C]. Similar detection limits are attainable using the TD-700 fluorometer. In these assays, reaction mixtures (100 µL of 1X Digestion Buffer and 100µL of BODIPY casein) were incubated in a 96-well microplate for one hour at room temperature, protected from light. The fluorescence was then measured in a fluorescence microplate reader. Footnotes [A] The Digestion Buffer (pH 7.8) is recommended for detecting the protease activity of most physiological enzymes with activity optima from pH 7.4 to 7.8. However, if you are working with an enzyme that requires the presence of specific co-factors or different pH conditions, a customized digestion buffer with the specific characteristics required should be used instead. The fluorescence of BODIPY FL-labeled protease hydrolysis products should be insensitive to pH in the range 3-10. [B] The sensitivity of the assay may be increased by incubating for longer time periods (up to 24 hours). For example, under conditions where 0.5 ng/mL trypsin produced an 8% fluorescence increase after incubation for 1 hour, the increase after 17 hours was 37%. For 50 ng/mL trypsin, the fluorescence increase was 69% after 1 hour and 132% after 17 hours. It is advisable to always run a reagent blank control sample (1.0 mL substrate working solution + 1.0 mL digestion buffer) incubated for the same length of time to account for any increase in background fluorescence. [C] Enzyme activity may vary depending on incubation buffers and temperature, as well as the storage conditions and number of freeze-thaw cycles to which the enzyme preparation has been subjected.
Warnings and Precautions BODIPY fluorophores, reactive dyes and their conjugates are covered by U.S. Patent Nos. 4,774,339; 5,187,288; 5,248,782; 5,274,113; and other U.S. and foreign patents pending. BODIPY and EnzChek are trademarks of Molecular Probes, Inc. All names containing the designation ® are registered with the U.S. Patent and Trademark Office. References
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