N-benzoyl-D-L-arginine-p-nitroanilide (BApNA), bovine serum albumin (BSA), bovine pancreatic trypsin, Trypsin-chymotrypsin inhibitor from Glycine max (SBBI) and Chitin from shrimp shells were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents used were of analytical grade.
Bovine pancreatic trypsin
Manufactured by Merck Group
Sourced in United States, Germany
Bovine pancreatic trypsin is a serine protease enzyme derived from the bovine pancreas. It is commonly used in laboratory settings for its ability to cleave peptide bonds, particularly those involving the carboxyl group of lysine or arginine residues. The core function of this enzyme is to facilitate protein digestion and sample preparation for various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Pd 10 column, by GE Healthcare (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (2 mentions)
Azocasein, by Merck Group (2 mentions)
Hyaluronidase h3506, by Merck Group (2 mentions)
Hydrochloric acid (hcl), by Merck Group (2 mentions)
Porcine pancreatic elastase, by Merck Group (2 mentions)
Bovine pancreatic α chymotrypsin, by Merck Group (2 mentions)
Trypsin chymotrypsin inhibitor from glycine max, by Merck Group (1 mentions)
Chitin from shrimp shells, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Acrylamide, by Merck Group (1 mentions)
L cysteine, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Bis acrylamide, by Merck Group (1 mentions)
Nα benzoyl d, by Merck Group (1 mentions)
Staphylococcus aureus, by American Type Culture Collection (1 mentions)
Pseudomonas aeruginosa, by American Type Culture Collection (1 mentions)
4 dimethylaminocinnamaldehyde, by Merck Group (1 mentions)
Proteinase k, by Merck Group (1 mentions)
22 protocols using bovine pancreatic trypsin
1
Trypsin Inhibition Assay Protocol
2
Purification and Modification of Alpha-2-Macroglobulin
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A2M was purified from plasma from a healthy volunteer using established protocols (27 (link), 28 (link)) and was never frozen. HEPES-buffered saline (HBS; 20 mM HEPES-NaOH, 150 mM NaCl, pH 7.4) was used as the running buffer for SEC and as the base buffer for all experiments, unless noted otherwise.
To prepare A2M-MA, purified A2M from plasma was treated with 0.5 M of methylamine, pH eight at 37 °C for 24 h, followed by desalting into HBS on a PD-10 column (GE Healthcare). To prepare A2M-T, bovine pancreatic trypsin (Sigma-Aldrich) was added to A2M at a 2.4:1 protease:A2M M ratio and incubated for 2 min at 37 °C. Trypsin was then inhibited by 2 mM phenylmethanesulfonyl fluoride (PMSF) at room temperature for 15 min. Excess trypsin and PMSF were removed by SEC on a Sephacryl S-300 HR column.
To prepare deglycosylated A2M, A2M was incubated overnight at room temperature with a 1:10 w/w ratio deglycosidase:A2M of bacterially expressed PNGase F, N-glycosidase F, Endo F2, and Endo F3 (29 (link)), which were then removed by SEC. Deglycosylated A2M-MA and A2M-T were prepared from deglycosylated native A2M as described above.
To prepare A2M-MA, purified A2M from plasma was treated with 0.5 M of methylamine, pH eight at 37 °C for 24 h, followed by desalting into HBS on a PD-10 column (GE Healthcare). To prepare A2M-T, bovine pancreatic trypsin (Sigma-Aldrich) was added to A2M at a 2.4:1 protease:A2M M ratio and incubated for 2 min at 37 °C. Trypsin was then inhibited by 2 mM phenylmethanesulfonyl fluoride (PMSF) at room temperature for 15 min. Excess trypsin and PMSF were removed by SEC on a Sephacryl S-300 HR column.
To prepare deglycosylated A2M, A2M was incubated overnight at room temperature with a 1:10 w/w ratio deglycosidase:A2M of bacterially expressed PNGase F, N-glycosidase F, Endo F2, and Endo F3 (29 (link)), which were then removed by SEC. Deglycosylated A2M-MA and A2M-T were prepared from deglycosylated native A2M as described above.
3
Extraction and Antimicrobial Evaluation of Jatropha curcas Seed Cake
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Jatropha curcas seed cake was obtained from Instituto Fazenda Tamanduá (Paraíba, Brazil), grounded in a coffee grinder and passed through a 1-mm-mesh screen. The resulting flour was treated with n-hexane (1:5, m/v) to remove the remaining oil left after biodiesel extraction. Defatted flour was stored in air-tight containers at 4°C until analysis. The bacteria Salmonella enterica subspecies enterica serovar choleraesuis (ATCC 10708), Bacillus subtilis subspecies spizizenii (ATCC 6633), Pseudomonas aeruginosa (ATCC 25619), and Staphylococcus aureus (ATCC 25923) were obtained from the Department of Biology (UFC), Fortaleza, Brazil. Swiss mice (Mus musculus), 20–25 g, were from the animal house at UFC. Azocasein, bovine pancreatic trypsin, bovine pancreatic chymotrypsin, bovine serum albumin (BSA), soybean trypsin inhibitor (SBTI), Nα-benzoyl-D,L-arginina-p-naftilamida (BANA), 4-(dimethylamino)cinnamaldehyde (DMACA), ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), L-cysteine and Nα-benzoyl-D,L-arginine 4-nitroanilide hydrochloride (BAPNA), sodium dodecyl sulfate (SDS), molar mass markers, acrylamide, bis-acrylamide, dithiothreitol (DTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chromatographic matrixes were from GE Healthcare. All other chemicals and reagents used were of analytical grade.
4
Collagen Fibril Diameter Analysis in Murine Spinal Discs
5
Serine Protease Inhibition Assay
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Protease inhibition assays were performed with purified recombinant NvKSPI-1 and NvKSPI-2 proteins using the method described by Ling et al. [56 (link)]. Three typical serine proteases (bovine pancreatic trypsin, bovine pancreatic chymotrypsin, and proteinase K, 200 ng/mL) (Sigma, Taufkirchen, Germany) and their corresponding substrates (N-benzoyl-Val-Gly-Arg-p-nitroanilide, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilid, and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilid) (Sigma, Taufkirchen, Germany) were selected for determination of the spectrum of enzyme inhibition by recombinant NvKSPI-1 and NvKSPI-2 as follows. The recombinant NvKSPI-1 and NvKSPI-2 (1 µg each) were pre-incubated with a reaction buffer (100 mM Tris-HCl, 100 mM NaCl, 1 mM CaCl2, pH 7.5) containing 200 ng/mL serine protease for 30 min at room temperature, and then 200 μL substrate was added (0.1 mM, 100 mM Tris-HCl, 100 mM NaCl, 1 mM CaCl2, pH 7.5) before measuring the absorbance at 405 nm every min for 30 min. Substrates alone were used as blanks. Buffer and buffer with bovine serum albumin (BSA) were used as controls. One unit of enzyme activity was defined as an increase of absorbance by 0.001 per min. The experiments were repeated three times as independent biological replicates.
6
Isolation and Characterization of Mammalian Reovirus
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The fecal supernatants that contained both L1 and S1 genes-positive RNAs were inoculated
onto monolayers of Madin-Darby Canine Kidney (MDCK) cells in maintenance medium (MM)
[32 (link)] supplemented with 2 μg/mL of bovine
pancreatic trypsin (Sigma, St. Louis, MO, USA). The cells were cultured at 37°C in 5%
CO2 and observed daily to trace the development of cytopathic effects (CPE).
The infected cell cultures that showed CPE were frozen and thawed three times, then
centrifuged at 1,220 × g for 15 min at 4°C, and the culture supernatants
were aliquoted and preserved at −80°C. Two more serial passages were carried out using the
supernatants from the previous passage into fresh monolayers. The supernatants from the
third passages were tested for the prevalence of MRVs L1 gene by RT-PCR.
The virus was subjected to three consecutive plaque purifications by plaque assay, as
described previously [13 (link)]. The plaque from the
third plaque cloning was tested to verify that the virus was a plaque-purified strain of
MRVs, based on RT-PCR with the use of S1 gene primer pairs as described above.
onto monolayers of Madin-Darby Canine Kidney (MDCK) cells in maintenance medium (MM)
[32 (link)] supplemented with 2 μg/mL of bovine
pancreatic trypsin (Sigma, St. Louis, MO, USA). The cells were cultured at 37°C in 5%
CO2 and observed daily to trace the development of cytopathic effects (CPE).
The infected cell cultures that showed CPE were frozen and thawed three times, then
centrifuged at 1,220 × g for 15 min at 4°C, and the culture supernatants
were aliquoted and preserved at −80°C. Two more serial passages were carried out using the
supernatants from the previous passage into fresh monolayers. The supernatants from the
third passages were tested for the prevalence of MRVs L1 gene by RT-PCR.
The virus was subjected to three consecutive plaque purifications by plaque assay, as
described previously [13 (link)]. The plaque from the
third plaque cloning was tested to verify that the virus was a plaque-purified strain of
MRVs, based on RT-PCR with the use of S1 gene primer pairs as described above.
7
Synthesis and Characterization of Peptides
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Human plasma αT (EC 3.4.21.5) and ProT were purchased from Haematologic Technologies (Essex Junction, VT, USA). Ecarin from Echis carinatus venom, bovine pancreatic trypsin, human plasma fibrinogen, Ac-Tyr-NH2, Ac-Phe-NH2, fluorescein isothiocyanate, and PABA were purchased from Sigma (St. Louis, MO, USA). The chromogenic substrate S2238 was from Chromogenix (Milan, Italy). SPAN12 monoclonal antibody was purchased from Beckman Coulter (Brea, CA, USA). Hirugen (54GDFEEIPEEY*LQ65) and [F]-hirugen58 (link), fibrinogen γʹ–peptide (408VRPEHPAETEY*DSLY*PEDDL427)63 (link), PAR1(38–60) (38LDPR↓SFLLRNPNDKYEPFWEDDE60)57 (link), Hir(1–47)51 (link), and αSyn(103–140) were synthesized by standard solid phase Nα-fluorenylmethyloxycarbonyl chemistry on a PS3 automated synthesizer (Protein Technologies Int., Tucson, AZ, USA), purified to homogeneity (> 98%) by semipreparative RP-HPLC, and characterized by high resolution mass spectrometry. Notably, Y* indicates phosphorylated Tyr residues. Nα-Fmoc-protected amino acids, solvents, and reagents for peptide synthesis were purchased from Applied Biosystems (Forster City, CA, USA) or Bachem AG (Bubendorf, Switzerland). Salts, solvents, and other reagents were of analytical grade and purchased from Sigma or Fluka (Darmstadt, Germany).
8
Quantifying Cecal and Fecal Trypsin Activity
9
In Vitro Gastric and Duodenal Digestion
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To reproduce gastric digestion in vitro, PWG gliadin was suspended in 0.03 N hydrochloric acid and incubated at 37 °C for 10 min with stirring. Pepsin (Sigma Aldrich, St Louis, MO, USA) was added and incubated at 37 °C for 60 min with stirring. The reaction was then stopped by incubating gliadin in a dry bath at 95 °C for 5 min to inactivate the enzyme. Gastric digests were adjusted to pH 6.0 with sodium phosphate buffer and subjected to simulated duodenal digestion by sequential addition of bovine pancreatic trypsin (Sigma Aldrich, St Louis, MO, USA) and type II bovine pancreatic-α-chymotrypsin (Sigma Aldrich, St Louis, MO, USA) at 37 °C for 30 min. The reaction was then stopped by introducing PWG gliadin in a dry bath at 95 °C for 5 min [37 (link)].
10
Biomaterials for Cell Culture Applications
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Polyvinyl alcohol (molecular weight (MW) 124,000–184,000 Da), 70% hydrochloric acid, acetonitrile, elementary bromine, perchloric acid, sodium bicarbonate, bovine pancreatic trypsin, poly-L-lysine, Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum, penicillin/streptomycin solution, gentamicin solution, Heregulin β1, Forskolin, ethylenediaminetetraacetic acid, [3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide] and dialysis membranes (cut-off 8000 Da) were purchased from Sigma-Aldrich (S. Louis, Missouri, USA). Potassium permanganate, ascorbic acid, acetic acid, 2-propanol, 100% ethanol, sodium iodide, sodium cyanoborohydride and dimethyl sulfoxide were purchased from Carlo Erba (Milan, Italy).
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