On the basis of hydrolysis of fluorogenic succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (suc-LLVY-AMC) peptides, Boc-Leu-Arg-Arg-AMC (Boc-LRR-AMC) and Z-Leu-Leu-Glu-AMC (Z-LLE-AMC), the chymotrypsin-like, trypsin-like and caspase-like activity of proteasomes were measured to determine their proteolytic activity. The hydrolysis assay of a fluorogenic substrate for proteasome activity was carried out using purified proteasome and 12.5 μM of a fluorogenic substrate (Enzo Life Sciences) in assay buffer (50 nM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mg ml−1 BSA, 1 mM ATP, 1 mM DTT). Relative fluorescence unit was measured after a 30 min incubation at room temperature. To measure proteasome activity in cells, whole-cell extracts were prepared in lysis buffer (100 mM NaCl, 50 mM NaH2PO4 (pH 7.5), 10% Glycerol, 5 mM MgCl2, 0.5% NP40, 1 mM ATP, 1 mM DTT) containing protease inhibitors, homogenized using 10 strokes with 1 ml syringe (needle: 26G × 1/2″) and centrifuged to remove insoluble matter. The purified and whole-cell proteasome activities were monitored by measuring free AMC fluorescence on a black 96-well plate using a TECAN infinite m200 fluorometer (TECAN, Männedorf, Switzerland).
Fluorogenic substrate
Manufactured by Enzo Life Sciences
Fluorogenic substrate is a type of laboratory equipment used in various analytical and research applications. It is a chemical compound that emits fluorescent light upon enzymatic cleavage, allowing for the detection and quantification of specific enzymatic activities. The core function of a fluorogenic substrate is to provide a sensitive and reliable method for measuring enzyme activity in biological samples.
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3 protocols using fluorogenic substrate
1
Quantifying Proteasome Proteolytic Activity
2
MMP-9 Enzymatic Activity Assay
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The SensoLyte 520 MMP-9 Assay Kit (Ana Spec, Inc., #71155, Fremont, CA) was used to determine the enzymatic activity of MMPs with and without oligonucleotides. Purified MMP protein was added at the indicated concentrations in MMP Dilution Buffer (50 mM Tris–HCl, 200 mM NaCl, 5 mM CaCl2, 1 µM ZnCl2, 0.05% Brij-35, and 0.05% NaAzide, pH 7.0) with the addition of the indicated concentrations of oligonucleotides. Hundred microliters of hMMP9/oligonucleotide samples were supplemented with 1 µM ZnCl2 and 2 mM APMA, mixed, added to a black 96-well plate, and incubated at 37°C for 1 h. APMA was omitted from the reaction when assaying for the catalytic domain — MMP9 since preactivation was not required. After incubation, a final concentration of 20 µM of the fluorogenic substrate (Enzo Life Sciences) in 100 µl of 2× assay buffer (100 mM HEPES, 20 mM CaCl2, and 0.1% Brij-35, pH 7.0) was added to the activated MMP protein and reaction kinetics were quantified using a Synergy 2 microplate reader (BioTek, Inc., Winooski, VT) with an excitation wavelength/bandwidth of 360/40 and an emission wavelength/bandwidth of 460/40 at 37°C for 1 ½ h. Unless otherwise indicated, the final concentration of MMP9 protein was 2 nM with variable concentrations of ssDNA in the final assay reaction.
3
MMP-9 Activity Assay of Glycosylation Mutants
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