OMNIMMP^® RED fluorogenic substrate

Manuals And Inserts

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Specific Protocol

Guidelines for use of OMNIMMP^® RED fluorogenic substrate
Dissolve the peptide with DMSO for a 1 mM stock solution. To ensure accurate concentration is achieved when dissolving peptides, both peptide purity and content need to be taken into account. Here is an example calculating the amount of DMSO needed to dissolve 0.1 mg Prod. No. BML-P277 to 1 mM, when its purity is 93% and content is 75%: (mol/1910.7 g) x (1x103 mmol/mol) x (L/1 mmol) x (1x106 μl/L) x (g/1x103mg) x (0.75x0.1 mg) x (0.93)=36.5 μl DMSO. Store at -20°C in aliquots; DMSO stocks are stable for 3-6
months at -20°C.

1. Prepare 10X assay buffer: 500mM HEPES, 100mM CaCl2, 0.5% Brij-35, pH to 7.0. For MMP-2 add 100µM ZnCl2 to the 10X buffer, and substitute 500mM MOPS for HEPES. For MMP-3, substitute 500mM MES (2-[N-morpholino]ethane-sulfonic acid) for HEPES and pH to 6.0.
2. Because optimal amounts of peptide and MMP will vary, it is best to initially use a range of both. Suggested concentration ranges are 0.1-1µM peptide (depending on application, enzyme, and other factors), and 0-50nM MMP. In the end, initial velocity should be linear with respect to enzyme concentration, and the peptide concentration must be well below Km for continuous assays or above Km for endpoint assays. NOTE: vigorously mix intermediate aqueous dilutions immediately prior to use, as aqueous solubility is limited, but DMSO, which inhibits MMP activity, must be kept below 1% in the assay.
3. To calibrate the fluorometer, it is recommended that a fluorogenic control (e.g. 6’-TAMRA) be used in the following manner: Prewarm assay buffer to the reaction temperature in 2 or 3 wells in the microplate, then add the substrate peptide #BML-P277 to the concentration to be used in the assay, and mix. Once the fluorescent signal is constant, use this reading as the zero value. Using the same wells, with their mixtures of substrate peptide and buffer, add fluorogenic control at 2 or 3 final molar concentrations ranging between 2 and 10% of the substrate peptide molar concentration, such that the volume in the well matches that of the assay to be run. Read fluorescence. Use these values to build a standard curve relating fluorescence intensity to concentration of fluorogenic control. If multiple concentrations of substrate peptide are used, such as in Km determinations, this procedure must be performed for each substrate concentration, due to absorptive quenching by the substrate peptide.
4. Reactions (diluted assay buffer, enzyme, and substrate) are monitored in a fluorometer using an exitation filter of 545nm (540nm is sufficient) and detection filter of 576 nm (590 nm is sufficient; with cutoff, if available, set at 570 nm) at 25-37°C. Either cuvette or microplate (using black NBS microplates) format can be used. The reaction can be stopped with 50mM EDTA if desired. If MMP inhibitors are being used, incubate MMP in buffer with inhibitor for 30-60 min. prior to assay.

NOTE: This serves as a guide only. Exact assay conditions must be determined by the user. Related products also available from Enzo Life Sciences include recombinant and purified MMPs, MMP inhibitors, and MMP inhibitor screening kits.

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