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MMP-9 Fluorimetric Assay Kit

Cat.No.: Kit-3100
Product Overview: The MMP-9 Assay Kit provides a convenient assay for high throughput screening of MMP-9 inducers and inhibitors. It detects MMP-9 activity in a variety of biological samples using a 5-FAM/QXL520 fluorescence resonance energy transfer (FRET)5 peptide. In the intact FRET peptide, the fluorescence of 5-FAM is quenched by QXL520. Upon cleavage into two separate fragments by MMP-9 (Scheme 1), the fluorescence of 5-FAM is recovered, and can be monitored at excitation/emission wavelengths = 490 nm/520 nm. With excellent fluorescence quantum yield and longer wavelength, 5-FAM shows less interference from autofluorescence of test compounds and cellular components. Compared to an EDANS/DABCYL FRET substrate, this 5-FAM/QXL520 substrate provides better assay sensitivity. The assays are performed in a convenient 96-well or 384-well microplate format.
Description: Matrix metalloproteinases (MMPs) belong to a family of secreted or membrane-associated zinc endopeptidases capable of digesting extracellular matrix components.1,2 The importance of MMPs in tumor development and invasion as well as other diseases is well known. MMP-9, (92-kDa gelatinase, collagenase-IV)3,4 is involved in a number of diseases such as cancer, angiogenesis, alopecia, and metastasis. It is proposed as a therapeutic target for these diseases.
Storage: Store all kit Components at -20centigrade
Protect Components A and B from light and moisture
Components D and E can be stored at 4centigrade for convenience
Size: 100 Assays
Kit Components: Component A: MMP-9 substrate: 5-FAM/QXL520 FRET peptide, Ex/Em=490 nm/520 nm upon cleavage, 60uL
Component B: 5-FAM-Pro-Leu-OH, Fluorescence reference standard Ex/Em=490 nm/520 nm 1 mM, 10 uL
Component C: APMA, 4-aminophenylmercuric acetate 1 M, 20 uL
Component D: Assay buffer, 20 mL
Component E: Stop solution, 10 mL
Materials Required but Not Supplied: Purified MMP-9
96-well or 384-well microplate: Black, flat-bottom 96-well or 384-well plate with non-binding surface.
Fluorescence microplate reader: Capable of detecting emission at 520 nm with excitation at 490 nm
Features & Benefits: Convenient Format: All essential assay components are included.
Optimized Performance: Optimal conditions for the detection of MMP-9 activity.
Enhanced Value: Less expensive than the sum of individual components.
High Speed: Minimal hands-on time.
Assured Reliability: Detailed protocol and references are provided.
Assay Protocol: Protocol A. Screening protease inhibitors using purified or recombinant MMP-9
1. Activate pro-MMP-9.
1.1 Incubate pro-MMP-9 with 1 mM APMA (diluted Component C) for 2 h at 37°C. Activate pro-MMP-9 immediately before the experiment.
Note 1: Keep activated enzyme on ice. Avoid vigorously vortexing the enzyme. Prolonged storage of activated enzyme will further de-activate the enzyme.
Note 2: APMA can be diluted with assay buffer (Component D). APMA belongs to the organic mercury class of compounds and must be handled with care! Dispose according to appropriate regulations.
Note 3: Activation of zymogen by APMA at higher protein concentration is preferred. After activation, the enzyme may be further diluted.
Note 4: MMP-9 is catalytic domain enzyme which does not require pre-activation
2. Prepare working solutions.
Note: Warm all kit components until thawed to room temperature before starting the experiments.
2.1 MMP-9 substrate solution: Dilute MMP-9 substrate (Component A) 1: 100 in assay buffer (Component D).
Table 1. MMP-9 substrate solution for one 96-well plate (100 assays).
Components Volume
MMP-9 substrate (100X, Component A) 50 uL
Assay buffer (Component D) 4.95 mL
Total volume 5 mL
2.2 MMP-9 diluent: Dilute activated MMP-9 to an appropriate concentration in assay buffer (Component D).
3. Set up enzymatic reaction.
3.1 Add test compounds and MMP-9 diluent into microplate. The suggested total volume of MMP-9 diluent and test compound is 50 uL (96-well plate) or 20 uL (384-well plate).
3.2 Simultaneously set up the following controls.
Positive control contains MMP-9 diluent without test compound.
Inhibitor control contains MMP-9 diluent and a known MMP-9 inhibitor.
Vehicle control contains MMP-9 diluent and vehicle used in delivering test compound (e.g. DMSO).
Test compound control contains assay buffer and test compound. Some test compounds have strong autofluorescence and may give false results.
Substrate control contains assay buffer only.
3.3 Using the assay buffer (Component D), bring the total volume of all controls to 50 uL (96-well plate) or 20 uL (384-well plate).
4. Pre-incubation.
4.1 Incubate the plate at the desired temperature for enzymatic reaction for 10-15 min. Also incubate MMP-9 substrate solution at the same temperature.
5. Initiate the enzymatic reaction.
5.1 Add 50 uL (96-well plate) or 20 uL (384-well plate) of MMP-9 substrate solution to each well. Mix the reagents completely by shaking the plate gently for 30-60 sec.
5.2 Measure fluorescence signal:
For kinetic reading: Immediately start measuring fluorescence intensity at Ex/Em=490 /520 nm and continuously record data every 5 min for 30 to 60 min.
For end-point reading: Incubate the reaction at 37°C for 30 to 60 min. Keep plate from direct light. Optional: Add 50 uL (96-well plate) or 20 uL (384-well plate) per well of stop solution (Component E). Mix the reagents. Then measure fluorescence intensity at Ex/Em=490/520 nm.
5.3 Data analysis: Refer to Appendix I.

Protocol B. Measuring MMP-9 activity in biological samples
Note: The FRET substrate in this kit can also be cleaved by MMP-1, 2, 3, 7, 8, 12, 13, and 14. If several MMPs are coexisting in your samples and you want to specifically measure MMP-9’s activity, please choose the Plus 520 MMP-9 Assay Kit (Kit-3101). Alternatively, MMP-9 can be isolated by immunoaffinity purification or other methods first before measuring its specific activity using the current assay kit.
1. Prepare MMP-9 containing biological samples.
1.1 Collect synovial fluids or supernatant of cell culture media (e.g. stimulated fibroblast) and centrifuge for 10-15 min at 1,000X g, 4°C. Collect the supernatant and store at -70°C until use.
1.2 Tissue samples should be homogenized in assay buffer (Component D) containing 0.1% (v/v) Triton-X 100, and then centrifuged for 15 min at 10000x g at 4°C. Collect the supernatant and store at -70°C until use.
Note: Triton-X 100 is not provided.
2. Activate pro-MMPs.
2.1 Incubate the MMP containing-samples with APMA (Component C) at the final concentration of 1 mM in the assay buffer (Component D) for 2 h at 37°C. Activate MMP immediately before the experiment.
Note 1: Keep activated enzyme on ice. Avoid vigorously vortexing the enzyme. Prolonged storage will further de-activate the enzyme.
Note 2: APMA can be diluted with assay buffer (Component D). APMA belongs to the organic mercury class of compounds and must be handled with care! Dispose according to appropriate regulations.
3. Prepare working solutions.
Note: Warm all kit components until thawed to room temperature before starting the experiments.
3.1 MMP-9 substrate solution: Dilute MMP-9 substrate (Component A) 1: 100 in assay buffer (Component D).
Table 1. MMP-9 substrate solution for one 96-well plate (100 assays).
Components Volume
MMP-9 substrate (100X, Component A) 50 uL
Assay buffer (Component D) 5 mL
Total volume 5 mL
3.2 MMP-9 diluent: If you use purified MMP-9 as a positive control, then dilute MMP-9 to an appropriate concentration in assay buffer (Component D).
Note: Pro-MMP-9 needs to be activated by APMA at higher protein concentration, and then be diluted to a working concentration in assay buffer. Please refer to Step 2. Avoid vigorous vortexing of enzyme.
4. Set up the enzymatic reaction.
4.1 Add 50 uL/well (96-well plate) or 20 uL/well (384-well plate) of MMP-9 containing sample.
4.2 Set up the following control:
Substrate control contains assay buffer [50 uL/well (96-well plate) or 20 uL/well (384-well plate)].
Positive control contains MMP-9 diluent [50 uL/well (96-well plate) or 20 uL/well (384-well plate)].
5. Initiate the enzymatic reaction.
5.1 Add 50 uL/well (96-well plate) or 20 uL/well (384-well plate) of MMP-9 substrate solution to the sample and control wells. Mix the reagents by shaking the plate gently for 30 seconds.
5.2 Measure fluorescence signal:
For kinetic reading: Immediately start measuring fluorescence intensity at Ex/Em=490 /520 nm continuously and record data every 5 min for 30 to 60 min.
For end-point reading: Incubate the reaction at 37°C for 30 to 60 min. Keep plate from direct light. Optional: Add 50 uL/well (96-well plate) or 20 uL/well (384-well plate) of stop solution (Component E). Mix the reagents and measure fluorescence intensity at Ex/Em=490/520 nm.
5.3 Data analysis: Refer to Appendix I.
Analysis: Appendix I: Data Analysis
The fluorescence reading from the substrate control well represents the background fluorescence. Subtract this background reading from the readings of the other wells to get the relative fluorescence unit (RFU).
For kinetic reading:
Plot data as RFU versus time for each sample. To convert RFU to concentration of the product of enzymatic reaction, please refer to Appendix II for setting up the fluorescence reference standard.
Determine the range of initial time points during which the reaction is linear. 10-15% conversion appears to be the optimal range.
Obtain the initial reaction velocity (Vo) in RFU/min. Determine the slope of the linear portion of the data plot.
A variety of data analyses can be done, e.g., determining inhibition %, EC50, IC50, Km, Ki, etc.

For endpoint reading:
Plot data as RFU versus concentration of test compounds or enzyme concentration.
A variety of data analyses can be done, e.g., determining inhibition %, EC50, IC50, etc.

Appendix II: Instrument Calibration
5-FAM fluorescence reference standard: Dilute 1 mM 5-FAM-Pro-Leu-OH (Component B) to 5 uM in assay buffer (Component D). Perform 2-fold serial dilutions to get concentrations of 2.5, 1.25, 0.625, 0.3125, 0.156 and 0.078 uM, include an assay buffer blank. Add 50 uL/well (96-well plate) or 20 uL/well (384-well plate) of these serially diluted 5-FAM-Pro-Leu-OH reference solutions.
Add 50 uL/well (96-well plate) or 20 uL/well (384-well plate) of MMP-9 substrate solution (refer to Protocol A, step 2.1 for preparation).
Note: MMP-9 substrate solution should be added to the 5-FAM reference standard to correct for the absorptive quenching by the FRET peptide. If multiple concentrations of substrate are used, this step must be performed for each concentration.
If the stop solution (Component E) was added into the enzymatic reaction before taking the end-point reading, the same volume of stop solution should be added to the reference standard wells to obtain a better comparison.
Measure the fluorescence intensity of the reference standard wells at Ex/Em=490 nm/520 nm. Use the same setting of sensitivity as used in the enzyme reaction.
Plot 5-FAM fluorescent reference standard as RFU (relative fluorescent unit) versus concentration.
Note: The final concentrations of the 5-FAM-Pro-Leu-OH reference standard solutions are 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.039, and 0 uM. This reference standard is used to calibrate the variation of different instruments and to account for experimental variability. It is also an indicator of the amount of final product of the MMP-9 enzymatic reaction.

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