Fluorogenic Substrate Assay for MMP-3

Youssef Farhat, Written 12/7/12, Last updated 5/6/14

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Introduction

Fluorogenic substrates are effective tools for assessing the activity of proteases in media, cell or tissue lysates.  Proteases, such as MMPs, are scissor-like proteins whose job is to cut certain amino acid sequences that exist within other proteins, such as collagen.  Fluorogenic substrates (also known as FRET substrates) are designed with an amino acid sequence that is cleavable by the protease of interest.  They are also designed in such a way that when a protease cuts the substrate, a fluorescent dye gets released and starts emitting a fluorescent signal.  The amount of signal detected is therefore directly related to how much protease activity there is in a given sample.

Here, we will use a fluorogenic substrate that is specifically cleaved by MMP-3 and MMP-12.  Since MMP-12 is not present in my samples (tendon cells), the assay is cleaved exclusively by MMP-3 in my experiments.

Note 1: This protocol is preliminary and is currently being tested.  For now, it only serves as an example of how one might use these fluorogenic substrates in an experiment.  It will likely change over time.

Note 2: It is good practice to keep fluorescent chemicals such as the substrate used here covered and protected from light as much as possible.  This can be achieved by wrapping containers with aluminum foil and working with minimal lighting.

Materials

• 520 MMP FRET Substrate XIII Substrate (Anaspec, #60580-01)
• Assay buffer (I am using some leftover buffer from another assay kit – Anaspec, #71129).  The following recipe has been used by others to make assay buffer [1]
  • 50 mM Tris-HCl pH 7.6, 200 mM NaCl, 5 mM CaCl2, 20 μM ZnSO4 and 0.05% Brij-35

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• DMSO (Sigma-Aldrich, #D2650)
• Black, flat-bottomed 96-well plates (e.g. Dynex, #3010 or Greiner Cellstar, #655086)
• MMP-3 standard (Anaspec, #72006)
• Deionized water
• Typical lab equipment and supplies such as pipettors, micropipettors, pipette tips, Eppendorf tubes, and a plate reader

Procedure

1. Reconstitute and Aliquot the Fluorogenic Substrate

1. Prepare 1 mM DMSO solution by combining 25 mL of DI water and 1.78 µL of DMSO.
2. Reconstitute the fluorogenic substrate by adding 467 µL of 1 mM DMSO.  This should make the substrate at a concentration of 100 µM.
3. Vortex briefly to ensure the substrate dissolves completely, and then lightly centrifuge the droplet to the bottom of the tube.
4. Aliquot 50 µL of the substrate into 1.5 mL Eppendorf tubes and store at -20ºC.

2. Perform the Assay

1. Samples should be thawed and kept on ice.
2. Dilute the appropriate amount of fluorogenic assay to 2 µM as follows:
   1. Determine the total number of samples and standards that will be used in the assay, “S.”
   2. Multiply “S” by 50 to obtain volume, “V1,” in microliters.
   3. If you will use a normal micropipettor to add substrate to the samples, add 100 µL to “V1” in order to obtain “V2”.  If you will use a multichannel pipettor instead, add 1.5 mL to “V1” in order to obtain “V2.”
   4. Divide “V2” by 50.  This is the volume of 100 µM substrate that is needed.
   5. Multiply “V2” by 49/50.  This is the volume of Assay buffer needed.
   6. Combine the amounts of substrate and assay buffer determined above into the appropriate container (i.e. to reagent reservoir if using a multichannel pipettor).
   7. Protect the substrate from light by covering it with foil.
3. Add 50 µL of samples and standards to the appropriate wells of a black, 96-well plate
4. Set the plate reader to shake the plates for 15 seconds and then make fluorescence measurements with an excitation wavelength of 490 ± 9 nm and emission wavelength of 520 ± 9 nm.  The plate reader can be set to read every 5 minutes for 30 min-1 hour, or to read only once at any time between 30 min and 1 hour.
   • Sensitivity for the plate reader should be set to an appropriate value.  For our lab’s plate reader, I set sensitivity to 60 and found that it results in a baseline fluorescence reading of ~200 RFU for blank samples (the maximum measurable fluorescence of our plate reader is ~100,000 RFU, giving a good range for measurements).
5. Add 50 µL of substrate to the samples and measure samples in the plate reader as indicated above.

References

[1] Candelario-Jalil, E. et al. “Cyclooxygenase inhibition limits blood-brain barrier disruption following intracerebral injection of tumor necrosis factor-alpha in the rat.” J Pharmacol Exp Ther. 2007 Nov;323(2):488-98.

Citing this Protocol

Farhat, Y. “Fluorogenic Substrate Assay for MMP-3.” The Protocol Place. Dec 2012. Accessed on mm/dd/yyyy. <http://protocol-place.com/>