After immunoprecipitation, the beads containing FLAG-tagged OpdB were separated in a 2:1 ratio for AMC assay:WB analysis. The AMC assay was performed by incubating the beads in 50 mM phosphate buffer (pH8) supplemented with 10 mM EDTA and 2 mM DTT. After 30 minutes incubation at 37°C, 200 μM z-Arg-Arg-AMC (Bachem) was added. Fluorogenic leaving groups were detected as described for PREP. Western blot was performed for normalization purposes. The beads were resuspended in 1x sample buffer containing 1% β mercaptoethanol. After 10 min at 95°C, the samples were analyzed by western blotting.
Z arg arg amc
Manufactured by Bachem
Sourced in Germany, Switzerland
Z-Arg-Arg-AMC is a synthetic peptide substrate used in biochemical assays. It consists of the amino acid sequence Benzyloxycarbonyl-Arginine-Arginine-7-Amino-4-Methylcoumarin. This substrate can be cleaved by enzymes that recognize and hydrolyze the arginine-arginine bond, releasing the highly fluorescent 7-Amino-4-Methylcoumarin moiety.
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Lab products found in correlation
Z phe arg amc, by Bachem (2 mentions)
Infinite m200 fluorometer, by Tecan (1 mentions)
Nupage novex system, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti β actin, by Santa Cruz Biotechnology (1 mentions)
Iblot2, by Thermo Fisher Scientific (1 mentions)
Western lightening plus ecl, by PerkinElmer (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
5 protocols using z arg arg amc
1
Fluorogenic Enzymatic Activity Assay
2
Enzymatic Activity Assays of TrCB1
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The peptidase activity of TrCB1 forms processed as described above (0.45 μg TrCB1 per well, i.e., 70 nM) was assayed with two synthetic fluorogenic substrates (25 μM, Bachem): Z-Phe-Arg-AMC, (cathepsin L/B substrate) and Z-Arg-Arg-AMC (cathepsin B-selective substrate) in 50/100 mM citrate/phosphate buffer (CPB), 2 mM DTT, pH 3–8 (final volume 200 μl). The reactions were performed in 96-well black flat bottom plates (Nunc) using Infinite M200 fluorometer (TECAN) with excitation and emission wavelengths set to 360 and 465 nm, respectively. The release of AMC was measured at RT in a 30 min kinetic cycle at 2 min intervals.
An exopeptidase (peptidyl dipeptidase) activity assay using Bz-Gly-His-Leu as a substrate was performed with pepTrCB1 forms employing a modified protocol (Sajid et al., 2003 (link)). The pepTrCB1 forms (1 μg, 160 nM) were incubated for 15 min with the substrate in 50/100 mM CPB pH 4–5.5 containing 2 mM DTT (final volume 100 μl). Spontaneous reaction of the emerging free amino groups of His-Leu with fluorescamine (0.05 mg/ml) was monitored in a fluorometer set to 390/475 nm excitation/emission wavelengths during a 30 min kinetic cycle at 2 min intervals.
An exopeptidase (peptidyl dipeptidase) activity assay using Bz-Gly-His-Leu as a substrate was performed with pepTrCB1 forms employing a modified protocol (Sajid et al., 2003 (link)). The pepTrCB1 forms (1 μg, 160 nM) were incubated for 15 min with the substrate in 50/100 mM CPB pH 4–5.5 containing 2 mM DTT (final volume 100 μl). Spontaneous reaction of the emerging free amino groups of His-Leu with fluorescamine (0.05 mg/ml) was monitored in a fluorometer set to 390/475 nm excitation/emission wavelengths during a 30 min kinetic cycle at 2 min intervals.
3
Enzymatic Activities of Lysosomal Hydrolases
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Enzymatic activities of lysosomal hydrolases were determined from liver homogenates (see above) using standard procedures with colorimetric substrates (p-Nitrophenyl-α-D-mannopyranoside for α-mannosidase and p-Nitrophenyl N-acetyl-β-D-glucosaminide for β-hexosaminidase). Absorption of nitrophenolate was determined at 405 nm in a 96-well plate reader. For cathepsin B determination, protease inhibitors were omitted from the lysis buffer. Cathepsin B was determined using fluorescence substrate Z-Arg-Arg-AMC (Bachem, Weil am Rhein, Germany) according to Barrett (Barrett, 1980 (link)).
4
Kinetics of Cathepsin B Inhibition
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Example 2
Powdered lipidated inhibitor of Cathepsin B, NS-629, was dissolved in 0.1 M phosphate buffer, pH 6.0, containing 1 mM EDTA and 0.1% (v/v) PEG for final concentration of 0.05 μM. The kinetic reaction between Cathepsin B and its lipidated inhibitor was analyzed by continuous measurements of the loss of enzymatic activity at different concentration of inhibitor in the presence of fluorogenic substrate Z-Arg-Arg-AMC (AMC=7-amido-4-methylcoumarin) (Bachem). Inhibitor NS-629 in increasing concentrations (0.01-0.06 mM concentration), recombinant Cathepsin B (0.05 mM) and the dithiothreitole (DTT) (0.5 mM) were mixed in a plate with 0.1 M phosphate buffer, pH 6.0, containing 1 mM EDTA and 0.1% (v/v) PEG. After 15 minutes incubation at 37° C. the inhibition kinetics of Cathepsin B and NS-629 were determined. The reaction was started by the addition of 150 μl of Cathepsin substrate Z-Arg-Arg-AMC solution and the kinetics of substrate hydrolysis was monitored continuously during 10 min by a TECAN plate reader at excitation and emission wavelengths of 370 and 460 nm, respectively. As can be seen in
5
Fluorometric Assessment of Cathepsin Enzymes
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Cathepsin B, L and D activities were assessed fluorometrically using the following substrates: Z-Arg-Arg-AMC (Bachem, Bulbendorf, Switzerland), Z-Phe-Arg-AMC (Bachem) and 7-methoxycoumarin-4-acetyl-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys-DNP-D-Arg-amide, as described previously [32 (link)]. LAL was determined by using the pro-fluorescent substrate 4-methylumbelliferyl oleate [33 (link)]. Western blotting was used to assess changes in the protein expression of cathepsin B, L and LAMP-1, following lysis of the cells in water at 4°C, and electrophoresis (90 min, 130 V) using 4–12% bis-tris gels (Novex Nupage system, Life Technologies, Carlsbad, CA, USA) with protein transfer (20 V, 7 min) to a PDVF membrane (iBlot 2, Life Technologies). And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution). Proteins were visualized using a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA), following exposure of membranes to chemiluminescence reagents (Western Lightening Plus-ECL, Perkin Elmer, Waltham, MA, USA), with densitometry performed using ImageJ software (National Institutes of Health, USA).
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