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Omnimmp fluorogenic substrate

Manufactured by Enzo Life Sciences
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The OmniMMP fluorogenic substrate is a versatile tool used to measure the activity of matrix metalloproteinases (MMPs). It provides a convenient and sensitive method for quantifying MMP activity in various biological samples.

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Lab products found in correlation

Spectramax m2, by Molecular Devices (2 mentions) Mmp 2, by Enzo Life Sciences (2 mentions) Mca pro leu gly leu dpa ala arg nh2 acoh, by Enzo Life Sciences (2 mentions) Softmax pro software version 5, by Molecular Devices (1 mentions) Mmp 12, by Enzo Life Sciences (1 mentions)

6 protocols using omnimmp fluorogenic substrate

1

MMP Activity Assay in Macrophages

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BMDMs were lysed with 1% Triton X-100 in PBS and OmniMMP Fluorogenic Substrate (400 µM, BML-P126-0001, Enzo Life Sciences, Farmingdale, NY, USA) was added in the 1× Omni-buffer (50 mM HEPES, 10 mM CaCl2 in dH2O, pH 7.0). Fluorescence was measured at 2 min intervals from 0–300 min at an excitation of 320 nm and emission of 405 nm with a SpectraMax M2 and SoftMax Pro Software Version 5 (Molecular Devices).
Tillie R.J., Theelen T.L., van Kuijk K., Temmerman L., de Bruijn J., Gijbels M., Betsholtz C., Biessen E.A, & Sluimer J.C. (2021). A Switch from Cell-Associated to Soluble PDGF-B Protects against Atherosclerosis, despite Driving Extramedullary Hematopoiesis. Cells, 10(7), 1746.
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2

Fluorogenic Assay for MMP-2 and MMP-12 Inhibition

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MMP-2 and −12, and OmniMMP fluorogenic substrate were purchased from Enzo Life Sciences (Farmingdale, NY, USA). The assays were carried out in black 96-well plates (Corning). Each well contained a volume of 100 μL including buffer (50 mM HEPES, 10 mM CaCl2, 0.05% Brij-35, pH 7.5), human recombinant MMP (1.16 U/well of MMP-2, 0.007 U/well of MMP-12, Enzo Life Sciences), inhibitor (various concentrations, 1mM – 5 μM), and fluorogenic OmniMMP substrate (4 μM Mca-Pro-LeuGly-Leu-Dpa-Ala-Arg-NH2•AcOH, Enzo Life Sciences). The enzyme and inhibitor were incubated in solution at 37 °C for 30 min, followed by the addition of the substrate to initiate the reaction. The change in fluorescence was monitored over 20 min with excitation and emission wavelengths at 320 and 400 nm, respectively. The negative control wells, containing no inhibitor, were arbitrarily set as 100% activity. The positive control wells, containing 200 μM NSA, were arbitrarily set as 0% activity. MMP activity was defined as the ratio of fluorescence increase in the inhibitor wells relative to the negative control wells, expressed as a percentage. The assays were performed in triplicate.
Chen A.Y., Thomas P.W., Stewart A.C., Bergstrom A., Cheng Z., Miller C., Bethel C.R., Marshall S.H., Credille C.V., Riley C.L., Page R.C., Bonomo R.A., Crowder M.W., Tierney D.L., Fast W, & Cohen S.M. (2017). Dipicolinic Acid Derivatives as Inhibitors of New Delhi Metallo-β-lactamase-1. Journal of medicinal chemistry, 60(17), 7267-7283.
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3

Quantifying Endothelial MMP Activity

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MMP activity was measured from equal volumes of cell-free supernatant from confluent endothelial cells grown on transwell filters as described above. 3 h after incubation with OMNIMMP® fluorogenic substrate (Enzo) at 37 °C, fluorescence of the cleaved substrate was measured at an emission/excitation wavelength of 280/360 nm according to the manufactures’ protocol.
Storck S.E., Kurtyka M, & Pietrzik C.U. (2021). Brain endothelial LRP1 maintains blood–brain barrier integrity. Fluids and Barriers of the CNS, 18, 27.
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4

Fluorometric MMP-2 Activity Assay

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MMP-2 and OmniMMP fluorogenic substrate were purchased from Enzo
Life Sciences. The assays were carried out in black NUNC 96-well plates.
Each well contained a volume of 100 µL including buffer (50 mM
HEPES, 10 mM CaCl2, 0.05% (v/v) Brij-35, pH 7.5), human
recombinant MMP (1.16 U MMP-2, Enzo Life Sciences), inhibitor (100
µM), fluorogenic OmniMMP substrate (4 mM Mca-Pro-Leu
-Gly-Leu-Dpa-Ala-Arg-NH2·AcOH, Enzo Life Sciences). The mixture
of protein and inhibitor was pre-incubated in solution at 37
oC for 30 min, followed by the addition of the substrate
to initiate the reaction. The change in fluorescence was monitored for
30 min by BioTek synergy HT fluorescence plate readers with excitation
and emission wavelengths at 320 and 400 nm, respectively. The control
wells, containing no inhibitor, were arbitrarily set as 100% activity.
MMP activity was defined as the ratio of fluorescence increase in the
inhibitor wells relative to the negative control wells, expressed as a
percentage. The assays were performed in triplicate. The data were
normalized to values measured for uninhibited enzyme. Assay data are
reported as the mean±standard deviation.
Li J., Zhang Y., Da Silva Sil Dos Santos B., Wang F., Ma Y., Perez C., Yang Y., Peng J., Cohen S.M., Chou T.F., Hilton S.T, & Deshaies R.J. (2018). Epidithiodiketopiperazines inhibit protein degradation by targeting proteasome deubiquitinase Rpn11. Cell chemical biology, 25(11), 1350-1358.e9.
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5

Fluorometric Assay of MMP Activity

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The functional activity of matrix metalloproteinase (MMP) was determined using OmniMMP™ fluorogenic substrate (Enzo Life Science, BML-P126-0001). Fluorescence was detected at an interval of two minutes on a Spectromax (Ex 328 nm, Em 393 nm, Molecular Devices SPECTRAmax M2).
van Kuijk K., Demandt J.A., Perales-Patón J., Theelen T.L., Kuppe C., Marsch E., de Bruijn J., Jin H., Gijbels M.J., Matic L., Mees B.M., Reutelingsperger C.P., Hedin U., Biessen E.A., Carmeliet P., Baker A.H., Kramann R.K., Schurgers L.J., Saez-Rodriguez J, & Sluimer J.C. (2021). Deficiency of myeloid PHD proteins aggravates atherogenesis via macrophage apoptosis and paracrine fibrotic signalling. Cardiovascular Research, 118(5), 1232-1246.
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6

Measuring Endothelial MMP Activity

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MMP activity was measured from equal volumes of cell-free supernatant from confluent endothelial cells grown on transwell filters as described above. 3 hours after incubation with OMNIMMP ® fluorogenic substrate (Enzo) at 37°C, fluorescence of the cleaved substrate was measured at an emission/excitation wavelength of 280/360 nm according to the manufactures' protocol.
Storck S.E., & Pietrzik C.U. (2021). Brain endothelial LRP1 maintains blood brain-barrier integrity.
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