For in vitro duodenal digestion, 100 μL of the 120 min gastric digest was used as starting material. The pH of the digests was adjusted to 8.5 with 0.1 M NaOH and the following were added: 22.8 μL of 0.15 M Tris/HCl buffer (pH 8.5) and 4.17 μL of 0.25 M sodium taurocholate (Sigma-Aldrich) solution. The simulated duodenal digestion was conducted at 37°C using bovine pancreas trypsin (Sigma-Aldrich) at an enzyme:substrate ratio of 1:2.8 (w/w). Aliquots were taken at 0, 60, and 120 min.
Bovine pancreas trypsin
Manufactured by Merck Group
Sourced in United States, Germany, Hungary
Bovine pancreas trypsin is a proteolytic enzyme derived from the pancreas of cattle. It is used in various laboratory applications that require the hydrolysis of peptide bonds in proteins.
Automatically generated - may contain errors
Lab products found in correlation
Sodium taurocholate, by Merck Group (1 mentions)
Alpha 1 antitrypsin, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Milli q water filtration system, by Merck Group (1 mentions)
K2so4, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Sytox blue dead cell stain, by Thermo Fisher Scientific (1 mentions)
Soybean trypsin inhibitor sbti, by Merck Group (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Superdex 200 hr 10 30 column, by Cytiva (1 mentions)
Image lab software 5, by Bio-Rad (1 mentions)
Trans blot sd semi dry electrophoretic transfer cell, by Bio-Rad (1 mentions)
N benzoyl l arginine ethyl ester baee, by Merck Group (1 mentions)
Folin ciocalteu s phenol reagent, by Merck Group (1 mentions)
8 protocols using bovine pancreas trypsin
1
In vitro Duodenal Digestion Protocol
2
MMP Activation Using Bovine Trypsin
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As an alternative for the highly toxic APMA, bovine pancreas trypsin (Sigma-Aldrich, St. Louis, MO, USA) was used for the activation of the MMPs. MMP2 was found to be degraded by trypsin treatment, but showed sufficient auto-activity in our assays. MMPs were activated at a concentration of 100 µg/mL in assay buffer (buffer as described, except for MMP14, the buffer of which consists of 50 mM Tris, 3 mM CaCl2, 1 µM ZnCl2, pH 8.5) for different times (see Table S1 ), with a final concentration of 5 µg/mL or 10 µg/mL trypsin. Afterwards, trypsin was inactivated with either or a combination of alpha-1 antitrypsin (final concentration of 100 µg/mL, Sigma-Aldrich), soybean trypsin inhibitor (SBTI, final concentration of 100 µg/mL, Sigma-Aldrich), or 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich). During all assays, controls for the effect of possible residual trypsin activity were performed and these were excluded. ADAM10 and ADAM17 did not require trypsin activation.
3
Bovine Trypsin Enzymatic Assay
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Bovine pancreas trypsin and ethanol were purchased from Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany. K2SO4, which was used to prepare saturated salt solution to maintain constant humidity in the desiccator, was obtained from Merck, Darmstadt, Germany. Water used to prepare salt solutions was of Millipore quality produced by MilliQ water filtration system from Merck, Darmstadt, Germany with a resistivity of 18 MΩ.cm at 25 °C.
4
Crystallization of Bovine Trypsin and Gal-3 Protein
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A 60 mg ml−1 sample of bovine pancreas trypsin (Sigma T7309) was prepared in 10 mg ml−1 benzamidine (VWR-Amresco Life Science), 3 mM calcium chloride and 20 mM Tris buffer at pH 8.0. Room-temperature crystals of various size and number were grown by varying 15–40%(w/v) PEG 4000 in 5% increments and 20–35%(w/v) ethylene glycol in 5% increments across a 24-well sitting-drop tray (HR3-160-Cryschem plate). After addition of 0.2 M Li2SO4 and 0.1 M 2-(N-morpholino)ethanesulfonic acid at pH 6.5 to each well, the total well buffer volume was brought to 1.0 ml with deionized water. Once the crystals grew, 10 µl of the well buffer from the next well to the right (3–5% higher concentration of PEG 4000) was added and mixed gently without breaking the crystals to ensure that the crystals were not stuck to the plastic. Crystals of Gal-3 protein in complex with a FAb (unpublished) were obtained using 10 mg ml−1 of the protein in buffer with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 50 mM NaCl, 1 mg ml−1 collagenase utilizing a hanging-drop method with equal-volume addition of the well solution containing 0.1 mM HEPES pH 7.5, 20% PEG 4000 and 10% 2-propanol.
5
Quantifying Bacterial Complement Deposition
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Bacteria were labelled with C5b-7 as described previously and subsequently washed three times by centrifugation (11000 rcf for 2 minutes). Bacteria were resuspended into RPMI + 0.05% HSA, which was used as buffer for all other incubations. For experiments where C5b-7 was shaved with trypsin, C5b-7 labelled bacteria at OD600 = 0.05 (5x107/ml) were incubated with 10 μg/ml bovine pancreas trypsin (Sigma) for 20 minutes at 37°C. Next, 25 μg/ml soy bean trypsin inhibitor (sbti, Sigma) was added along with 10 nM C8 and 20 nM C9 for 20 minutes at 37°C. For experiments where C5b-9 was trypsinized, C5b-7 labelled bacteria were first incubated with 10 nM C8 and 20 nM C9 for 20 minutes at 37°C and then exposed to 10 μg/ml trypsin for 20 minutes at 37°C. In all experiments, 2.5 μM of Sytox Blue Dead Cell stain (Thermofisher) was added to the final incubation step of the experiment. Samples were diluted 10–20 times and subsequently analyzed in the BD FACSVerse flow cytometer for FSC, SSC, Sytox, GFP and Cy5 intensity.
6
Cry1Ac Insecticidal Toxin Bioassay
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Cry1Ac protoxin and activated toxin were kindly supplied by the Biotechnology Research Group, Institute of Plant Protection, Chinese Academy of Agricultural Science. For activation, Cry1Ac protoxin was incubated 2 h at 37°C with 1/25 ratio of bovine pancreas trypsin (Sigma), and the soluble trypsinized toxin was purified by a Superdex 200 HR 10/30 column (Amersham Biosciences) on a fast protein liquid chromatography (FPLC) system. H. armigera bioassays with Cry1Ac protoxin and activated toxin were performed with a diet incorporation procedure. Approximately 1–1.5 g artificial diets incorporated with various concentrations of Cry1Ac protoxin or toxin suspended in distilled water or equal volume of distilled water (control) was put in each well of 24-well plates. A single first instar larva was placed into each well, and 24 larvae were used for each treatment. Mortality was recorded after seven days, and all assays were replicated three times. Pooled data were subjected to statistical analysis; the concentration killing 50% of larvae (LC50) was calculated by probit analysis with the computer program POLO (LeOra Software, Berkeley, California 1987).
7
FADS2 Protein Susceptibility to Trypsin Digestion
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WT and mutant FADS2 proteins with or without incubation with a molar excess of FAD were used for testing the susceptibility to trypsin digestion. Samples (10 μg) were incubated with or without 0.015 mg/mL trypsin (bovine pancreas trypsin, T8003, Sigma-Aldrich) at 0°C in 40 mmol/L HEPES/Na and 5 mmol/L 2-ME (400 μL). Aliquots of 2.5 μg protein were sampled at different time points, and the reaction was stopped by the addition of 0.3 mg/mL of soybean trypsin inhibitor (T9003, Sigma-Aldrich). As an internal standard for the quantity of loaded protein in the gel, BSA (1 μg) was also added to the loading buffer solution. The products of the proteolysis reaction were separated by 12% SDS-PAGE and electro-transferred onto a nitrocellulose membrane with a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). The immobilized proteins were incubated for 3 hr with a 3,000-fold dilution of a polyclonal antiserum against FADS.13 (link) The bound antibodies were visualized with the aid of secondary anti-rabbit IgG antibodies conjugated with alkaline phosphatase (1:3,500 dilution). Quantitative evaluation of immuno-reactive protein bands was carried out by densitometric analysis with ImageLab software 5.1 (Bio-Rad).
8
Trypsin-Mediated Wine Phenol Analysis
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Bovine pancreas trypsin (EC 3.4.21.4), gallic acid and N-benzoyl-L-arginine ethyl ester (BAEE) were purchased from Sigma-Aldrich Company (Budapest, Hungary). Folin-Ciocalteu's phenol reagent was the product of Merck (Darmstadt, Germany). Red wines were purchased from commercially sources: La Bonita
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