Measurement of both MMPs’ actual specific activity was conducted in a microplate (Greiner Bio-One, Rainbach im Mühlkreis, Austria) which was previously coated using respective specific metalloproteinase antibodies (the same as in Western blot (WB) assays) [30 (link)]. The relevant sample of one hundred microliters was added to each well for the purpose of immobilization of the metalloproteinase. The microplate was incubated overnight at 4 °C. All redundant proteins were washed out using TBS-T buffer (50 mM Tris/HCl pH 7.4, 0.9% NaCl, 0.05% Tween 20). MMPs’ activity was measured in 100 μL of 50 mM Tris/HCl buffer, pH 7.5, containing 10 mM CaCl2, 150 mM NaCl, and 0.025% Brij 35 with 4 μM fluorogenic substrate (MCA-Pro-Leu-Ala-Cys(p-OMeBz)-Trp-Ala-Arg(Dpa)-H2)) (Cat#444258; Merck, Germany). The microplate was incubated at 37 °C for 1 h with gentle mixing. A total of 25 μL of 100 mM EDTANa2 was used to stop the reaction. The fluorogenic substrate degradation was measured by means of a multimode microplate reader (Tecan Infinite® 200 PRO, Männedorf, Switzerland) with wavelengths of excitation at 325 nm and emission at 393 nm. Degraded substrate quantity was calculated on the basis of the calibration curve prepared under the same conditions with 7-amino-4-methylcoumarin (Sigma-Aldrich; Saint Louis, MO, USA). The specific activity of MMPs was given in katals per kg of protein.
Mca pro leu ala cys p omebz trp ala arg dpa h2
Manufactured by Merck Group
Sourced in Germany
MCA-Pro-Leu-Ala-Cys(p-OMeBz)-Trp-Ala-Arg(Dpa)-H2) is a peptide-based lab equipment product. It functions as a substrate for enzymatic analysis.
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Lab products found in correlation
3 protocols using mca pro leu ala cys p omebz trp ala arg dpa h2
1
Quantifying Metalloproteinase Activity
2
Quantification of MT1-MMP Activity
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Assay of MT1-MMP activity was performed in a black 96-flat-bottom-well microplate (Greiner Bio-One, Austria), which was precoated with specific anti-human MT1-MMP antibody (R&D Systems, USA). 25 The extract samples (100 μL) were added to the wells for immobilization of the MT1-MMP, and the microplate was incubated overnight at 4°C. The unbound proteins were washed out with TBS-T buffer (50 mmol/L Tris-HCl [pH 7.4], 0.9% NaCl, 0.05% Tween-20). To measure MMP activity, 100 μL of 50 mmol/L Tris-HCl buffer [pH 7.5] containing 10 mmol/L CaCl 2 , 150 mmol/L NaCl and 0.025% Brij 35 26 with MCA-Pro-Leu-Ala-Cys(p-OMeBz)-Trp-Ala-Arg(Dpa)-H 2 (Merck, Germany) (4 μmol/L final concentration) as a fluorogenic substrate was used. The microplate was incubated at 37°C for 60 minutes with gentle shaking. The enzymatic reaction was stopped by the addition of 25 μL of 100 mmol/L EDTANa 2 . Fluorescence of the released 7-amido-4methylcoumarin (AMC) was read with a multimode microplate reader (Tecan Infinite® 200 PRO, Tecan, USA) at the excitation and emission wavelengths set at 325 and 393 nm, respectively. Measurements were standardized with AMC. One unit of enzyme activity (U) was defined as the amount of enzyme releasing 1 μmol of product (AMC) per minute at 37°C.
3
Fluorometric Assay for MMP-1 and MMP-13 Activity
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Actual specific activity of MMP-1 and MMP-13 was measured in black 96-well flat-bottom microplates precoated with the respective specific MMP antibody (the same antibodies that were used in Western blot analysis) [18] . The appropriate sample was added to each well for MMP immobilization. Microplates were incubated overnight at 4 ° C. All other proteins were washed out with TBS-T buffer (50 mM Tris/HCl, pH 7.4, 0.9% NaCl, 0.05% Tween 20). MMP activity was measured in buffer (50 mM Tris/ HCl, pH 7.5) containing 10 mM CaCl 2 , 150 mM NaCl, and 0.025% Brij 35 [19] with MCA-Pro-Leu-Ala-Cys (p-OMeBz)-Trp-Ala-Arg (Dpa)-H2 (Merck, cat. No. 444258) as a fluorogenic substrate (4 µM final concentration). Microplates were incubated at 37 ° C for 60 min with gentle shaking. The reaction was stopped by addition of 100 mM EDTA Na 2 . Degradation of the fluorogenic substrate was measured with a spectrophoto-fluorimetric/multimode microplate reader (Tecan Infinite ® 200 PRO; Tecan) with excitation and emission wavelengths set at 325 and 393 nm, respectively. The quantity of degraded substrate was calculated from the calibration curve prepared at the same condition with 7-amino-4-methylcoumarin (Sigma-Aldrich; cat. No. 257370) as a standard. MMP-specific activity was expressed in katals per kilogram of protein.
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