Culture samples were centrifuged to collect the supernatant at 3000× g for 5 min at 4 °C. EnCh and ChB activities from the supernatant were determined by using the Chitinase Assay Kit (Sigma-Aldrich, Saint Louis, MO, USA), using 10 μL of supernatant and 90 μL of a specific substrate solution (1 mg.mL−1 of p-nitrophenol-triacylchitotriose [pNP-(GlcNAc)3] or p-nitrophenol-diacetylchitobiose [pNP-(GlcNAc)2]). The mixtures were incubated at 37 °C for 30 min; optical density was read at 405 nm using a microplate reader, model Spectramax® plus 384 (Molecular Devices, Sunnyvale, CA, USA). The substrates [pNP-(GlcNAc)3] and [pNP-(GlcNAc)2] were used as substrates for EnCh and ChB, respectively. One unit (U) of EnCh and ChB was defined as the amount of each enzyme required to release 1.0 μmol of p-nitrophenol (pNP) from each substrate per minute.
Chitinase assay kit
Manufactured by Merck Group
Sourced in United States
The Chitinase Assay Kit is a laboratory tool designed to measure the activity of the enzyme chitinase. Chitinase is an enzyme that breaks down chitin, a structural component found in the cell walls of fungi and the exoskeletons of insects and crustaceans. The kit provides the necessary reagents and protocols to quantify chitinase activity in a variety of sample types.
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Lab products found in correlation
Spectramax plus 384, by Molecular Devices (1 mentions)
Filtermax f5, by Molecular Devices (1 mentions)
Pro q emerald 300 lipopolysaccharide gel stain kit, by Thermo Fisher Scientific (1 mentions)
Pierce cell surface protein isolation kit, by Thermo Fisher Scientific (1 mentions)
Rapigest sf surfactant, by Waters Corporation (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Synergy h4 hybrid reader, by Agilent Technologies (1 mentions)
Synergy mx, by Agilent Technologies (1 mentions)
Microtiter plate, by Greiner (1 mentions)
Cs0980, by Merck Group (1 mentions)
Multichannel pipette, by Eppendorf (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Enspire multimode reader, by PerkinElmer (1 mentions)
19 protocols using chitinase assay kit
1
Quantifying Chitinase Enzyme Activity
2
Chitinase Activity Assays Optimization
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Two activity assays were carried out. The first assay was performed by incubating 2.5 μg of rChi1 with 1.5 mg of chitin-azure substrate (Sigma-Aldrich cod. C3020-1G) for 3 h at 37 °C in DPBS buffer with 10 mM MgCl2 [24 (link)]. A second activity assay was carried out with the Chitinase Assay kit (Sigma-Aldrich cod. CS0980) in the presence of 2 μg of enzyme and 50 μg of one of three substrates, 4-Nitrophenyl N,N′-diacetyl-β-D-chitobioside, 4-Nitrophenyl N-acetyl-β-D-glucosaminide, or 4-Nitrophenyl β-D-N,N′,N″-triacetylchitotriose, according to the kit instructions. All the assays were performed in triplicate.
3
Fluorometric Chitinase Activity Assay
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Chitinolytic enzyme activity was determined fluorometrically for the 41 strains studied using the chitinase assay kit (CS0980) from Sigma-Aldrich/Merck, following the manufacturer’s instructions, and a multi-mode microplate reader (Filter Max F5, Molecular Devices). For specifics on sample preparation prior to endo-chitinase and exo-chitinase activity assays, please see Additional file 2 : Detailed methodology. In brief, enzymatic activities were measured as the release of 4-methylumbelliferone (4-MU) from various 4-MU labeled substrates. Exo-chitinase (EC 3.2.1.52) activities were detected using the substrates 4-methylumbelliferyl N-acetyl-β-D-glucosaminide and 4-methylumbelliferyl N,N′-diacetyl-β-D-chitobioside hydrate to detect N-acetyl-β-glucosaminidase (release of GlcNAc monomers) and chitobiosidase (release of GlcNAc dimers) activity, respectively. Endo-chitinase (EC 3.2.1.14) activity was detected using 4-methylumbelliferyl β-D-N,N′,N″-triacetylchitotriose as substrate (release of GlcNAc trimers). All assays were performed at substrate concentrations of 0.5 mg/mL and sample volumes of 10 μL. Further details on physical-chemical parameters used in the assays and registration of results are provided in Additional file 2 : Detailed methodology.
4
Isolation and Characterization of F. succinogenes
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All reagents used in this work were supplied by Sigma-Aldrich (Dorset, UK) with the highest purity available, unless otherwise stated. All solvents were supplied by Fisher Scientific (Loughborough, UK). Pierce® Cell Surface Protein Isolation kit was supplied by Thermo Fisher Scientific (cat number 89881, Rockford, USA). Lipopolysaccharide (LPS) extraction Kit was supplied by iNtRON Biotechnology (cat number 17141, Kyungki-Do, Korea). Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit was supplied by Thermo Fisher Scientific (cat number, P20495, Loughborough, UK). A 4-plex iTRAQ reagent multiplex kit was supplied by SCIEX (P/N 4352135, Redwood, CA, USA). RapiGestTM SF Surfactant was supplied by Waters (cat number 186001861, Milford, MA, USA). Glucose-1-phosphate colorimetric assay kit was supplied by BioVision, Biosciences (cat number K697-100, Cambridge, UK). Chitinase assay kit was supplied by Sigma-Aldrich (cat number CS0980, Dorset, UK). Sequencing Grade Modified Trypsin was supplied by Promega (cat number, V5111, Southampton, UK). F. succinogenes S85 (ATCC 19169), was kindly provided by Professor Paul Weimer (US Dairy Forage Research Centre, Madison, Wisconsin, USA).
5
Chitin-induced Chitinolytic Activity Analysis
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Cells of strain SBC82T grown in the liquid medium MA supplemented with 0.1% (w/v) amorphous chitin instead of fructose were collected by centrifugation and lysed by sonication. Chitinolytic activities of the resulting cell extracts were measured in a fluorimetric assay with 4-methylumbelliferyl (4-MU) derivatives using a Chitinase Assay Kit (Sigma, CS1030). The following substrates were used: 4-MU- β-D-N, N′, N″-triacetylchitotriose, 4-MU-diacetyl-β-D -chitobioside and 4-MU-N-acetyl-β-D -glucosaminide. One unit of activity was defined as the amount of enzyme required to release 1 nmol of 4-MU from the appropriate substrate per minute. Assays were performed at 25°C in 50 mM sodium citrate buffer (pH 5.0). The fluorescence of liberated 4MU was measured using the fluorimeter Fluorat-02-Panorama (Lumex, Russian Federation), with excitation at 360 nm and emission at 450 nm.
6
Chitinase Activity Assay of Pseudomonas sp.
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Pseudomonas sp. TXG6-1 was grown in 100 mL of basal medium containing 0.1% K2HPO4 and 0.05% MgSO4•7H2O (pH 7.0), and supplemented with 0.5–2% (w/v) of colloidal chitin as the sole carbon/nitrogen (C/N) source. The bacteria were cultured aerobically in a 250 mL Erlenmeyer flask at 30 °C for three days on a rotary shaker (150 rpm). After centrifugation (3,000 × g, 4 °C, 15 min), the supernatants were collected for measurement of chitinase activity.
Chitinase activity was measured using a chitinase assay kit (Sigma-Aldrich), according to the manufacturer’s instructions. The exochitinase (N-acetyl-β-glucosaminidase), chitobiosidase and endochitinase activities in the harvested culture medium were determined using p-NPGlcNAc, p-NP-(GlcNAc)2 and p-NP-(GlcNAc)3 as substrates, respectively. The release of p-nitrophenol was monitored at 405 nm. A molar extinction coefficient for p-nitrophenol of ε = 17,700 M−1cm−1 was used to calculate the product concentration. Protein concentrations were measured by the method of Lowry et al. (1951) (link) using bovine serum albumin as the standard.
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Pseudomonas sp. TXG6-1 was grown in 100 mL of basal medium containing 0.1% K2HPO4 and 0.05% MgSO4•7H2O (pH 7.0), and supplemented with 0.5–2% (w/v) of colloidal chitin as the sole carbon/nitrogen (C/N) source. The bacteria were cultured aerobically in a 250 mL Erlenmeyer flask at 30 °C for three days on a rotary shaker (150 rpm). After centrifugation (3,000 × g, 4 °C, 15 min), the supernatants were collected for measurement of chitinase activity.
Chitinase activity was measured using a chitinase assay kit (Sigma-Aldrich), according to the manufacturer’s instructions. The exochitinase (N-acetyl-β-glucosaminidase), chitobiosidase and endochitinase activities in the harvested culture medium were determined using p-NPGlcNAc, p-NP-(GlcNAc)2 and p-NP-(GlcNAc)3 as substrates, respectively. The release of p-nitrophenol was monitored at 405 nm. A molar extinction coefficient for p-nitrophenol of ε = 17,700 M−1cm−1 was used to calculate the product concentration. Protein concentrations were measured by the method of Lowry et al. (1951) (link) using bovine serum albumin as the standard.
7
Chitinolytic Activity Quantification by Colorimetric Assay
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Test was preformed using Chitinase Assay Kit (CS0980, Sigma-Aldrich, USA). Procedure was based on technical bulletin obtained from producer [30 ]. Four groups of samples were prepared on microtiter plate: (1) Blanc—40 μL of substrate A, B or C; (2) Standard—120 μL of standard solution (included in Assay Kit); (3) Test—36 μL of substrate A, B or C with 5h to final concentration of 16 μg/mL (4 μL of 5h at 160 mg/mL); (4) Control—36 μL of substrate A, B or C with 4 μL of 0.2 mg/mL chitinase. Plate was incubated for 30 min at 37 °C and then the reaction was stopped with stop solution form the Assay Kit. Absorbance at 405 nm was measured using Synergy H4 Hybrid Reader (BioTek Instruments, USA) [27 ]. Chitinolytic activity was calculated using formula:
where: —chitinolytic activity [U/mL]; —absorbance of test sample at 405 nm [-]; —absorbance of blank at 405 nm [-]; —p-nitrofenol concentration in standard solution [μmol/mL]; —final volume of samples in each test well (after addition of stop solution) [mL]; —enzyme dilution factor (here equal to 1—enzyme was not diluted); —absorbance of standard [-]; —reaction duration time [min]; —volume of5h or chitinase [mL] (here 0.004 mL).
where: —chitinolytic activity [U/mL]; —absorbance of test sample at 405 nm [-]; —absorbance of blank at 405 nm [-]; —p-nitrofenol concentration in standard solution [μmol/mL]; —final volume of samples in each test well (after addition of stop solution) [mL]; —enzyme dilution factor (here equal to 1—enzyme was not diluted); —absorbance of standard [-]; —reaction duration time [min]; —volume of
8
Chitinase Activity Assay in Endophyte 3A12
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In an initial experiment, chitinase activity in wild type strain 3A12 was measured using the Chitinase Assay Kit (CS0980, Sigma) according to manufacturer recommendations. 4-nitrophenyl N,N′-diacetyl-β-D -chitobioside and 4-nitrophenyl N-acetyl-β-D -glucosaminide were used as enzyme substrates. Endophyte 3A12 was cultured in LB media for 2 days at 37°C and shaking at 250 rpm. Ten microliters of the 3A12 culture (OD595 = 0.8) or the chitinase control enzyme were used. The blank treatment consisted of 90 μl of the substrate with 10 μl of LB media. To measure whether chitinase activity is induced by S. homoeocarpa, 3A12 was cultured with and without the fungus, then chitinase activity was measured using 4-nitrophenyl N-acetyl-β-D -glucosaminide as a substrate. The blank treatment consisted of 90 μl of the substrate with 10 μl of LB media (with and without the fungus). Each assay was performed in triplicate, and each experiment was replicated. In a second experiment, chitinase activity was measured in the candidate mutants in triplicate using 4-nitrophenyl N-acetyl-β-D -glucosaminide as a substrate and compared to wild type, following the same protocol as noted above. For both experiments, the chitinase activity was calculated according to the following formula:
9
Stability and Chitinase Activity of DOI
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For the acid and alkali stability tests, DOI (10 nM) was treated with HCl (0.01, 0.1, and 1 M) or NaOH (0.01, 0.1, and 1 M) and incubated at 4 °C for 30 min to determine acid stability and alkali stability, respectively. The mixtures were then neutralized with either NaOH or HCl. The acid- or alkali-treated DOI was then added to the granulosa cells and incubated for 12 h. The cell culture medium was collected for the measurement of estradiol concentrations. For the thermal stability test, DOI was incubated at 60 °C, 80 °C, or 100 °C for 30 min. The heat-treated DOI was then added to the granulosa cells and incubated for 12 h. The cell culture medium was collected for the measurement of estrogen concentrations. The chitinase activity of DOI was measured using a Chitinase Assay Kit (# CS0980, Sigma-Aldrich) following the manufacturer’s instructions. Three substrates, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside (N6133), 4-nitrophenylN-acetyl-β-D-glucosaminide (9376), and 4-nitrophenyl β-D-N,N′,N′′-triacetylchitotriose (N8638) were used to evaluate the exochitinase activity (chitobiosidase activity), exochitinase activity (β-N-acetylglucosaminidase activity), and endochitinase activity of DOI, respectively.
10
Quantifying Chitinase Activity
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Chitinase activity was analysed using the Chitinase Assay Kit (Sigma-Aldrich) and carried out according to the manufacturer’s instructions. Briefly, cells induced for chitinase production during 4 h were resuspended to 0.0044 OD600 units/µl and 10 µl (0.044 OD600 units) were mixed with 10 µl 4-Nitrophenyl N,N’-diacetyl-β-d -chitobioside (1 mg/ml). Samples were incubated at 37 °C for 30 min, and the reaction was stopped by addition of 200 µl 39 mM sodium carbonate solution. Cells were harvested, and 200 µl supernatant was transferred to a microtiter plate (Greiner Bio-One) and the concentration of p-nitrophenol was measured as absorbance at 405 nm in a SynergyMx plate reader (BioTek). Specific activity was calculated as absorption per time per volume against a p-nitrophenol standard.
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