These plans are based on literature, please modify them to meet your specific needs.
1. Solution Preparation
(1) Preparation of stock solution: Dissolve Mca-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2 in DMSO to prepare the stock solution, with the concentration of 1-10mM.
Note: Unused stock solution should be aliquoted and stored in the dark at -20°C or -80°C to avoid repeated freezing and thawing.
(2) Prepare the working solution: Dilute the stock solution with PBS or an appropriate buffer at the working concentration of 1-10µM.
Note: Prepare the working solution fresh and store briefly at 4°C or on ice.
2. Detection of MMP activity in cell culture supernatant[1]
(1) Take 100µl of the cell culture supernatant and add Mca-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2 (final concentration of 10µM).
(2) Incubate at 37°C for 2 hours.
(3) Measure the fluorescence intensity at 405nm (excitation at 320nm) using an enzyme detector.
3. In vitro MMP activity assay[2]
(1) MMP enzymes at concentrations ranging from 0.5 to 20nM were pre-incubated at room temperature for 30 minutes, followed by the addition of a final concentration of 1µM Mca-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2.
(2) Incubation was conducted at 37°C for 2 hours.
(3) Fluorescence intensity at 405nm excitation, with a wavelength of 320nm, was measured using a microplate reader.
Precautions:
1) Fluorescent substrate has quenching problems. Please avoid light as much as possible to slow down fluorescence quenching.
2) For your safety and health, please wear a lab coat and disposable gloves.
References:
[1] Walsh, Logan A, and Sashko Damjanovski. “IGF-1 increases invasive potential of MCF 7 breast cancer cells and induces activation of latent TGF-β1 resulting in epithelial to mesenchymal transition.” Cell communication and signaling: CCS vol. 9,1 10. 2 May. 2011.
[2] Botkjaer, Kenneth A et al. “Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo.” Oncotarget vol. 7,13 (2016): 16773-92. |
