The enzymatic hydrolysis of the polymers was facilitated using the following procedure. In general, the polymers were cut into squares or rolled into balls of approximately 20 mg. Next, the samples were immersed into PBS buffer (1 ml, 100 mM), together with a protease from Bacillus licheniformis, (Sigma Aldrich, Zwijndrecht, The Netherlands); 1–2 mg of the protease enzyme was added to the polymer together with 40 µl of CaCl2 solution (0.01 M) at 37 °C under constant shaking. After 24 h, the residual samples were removed from the solution, dried in vacuo overnight at 40 °C, after which their residual weight was determined.
Protease from bacillus licheniformis
Manufactured by Merck Group
Sourced in United States
Protease from Bacillus licheniformis is a laboratory enzyme product. It is a proteolytic enzyme derived from the bacterial species Bacillus licheniformis. The core function of this product is to catalyze the hydrolysis of proteins into smaller peptides or amino acids.
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Lab products found in correlation
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
Coomassie brilliant blue r 250, by Merck Group (1 mentions)
Luria bertani broth, by Merck Group (1 mentions)
6 hydroxy 2 5 7 8 tetramethylchroman 2 carboxylic acid, by Merck Group (1 mentions)
Peptide digest assay standard, by Thermo Fisher Scientific (1 mentions)
Potassium peroxodisulfate, by Merck Group (1 mentions)
Iron 3 chloride hexahydrate fecl3 6h2o, by Merck Group (1 mentions)
2 4 6 tris 2 pyridyl s triazine, by Merck Group (1 mentions)
2 2 azino bis 3 ethylbenzothiazoline 6 sulfonic acid, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Aspergillus oryzae, by Merck Group (1 mentions)
90i microscope, by Nikon (1 mentions)
Alcalase, by Novozymes (1 mentions)
6 protocols using protease from bacillus licheniformis
1
Enzymatic Hydrolysis of Polymers
2
Extraction and Quantification of Garlic Compounds
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To reveal any possible peptide-bound alliin or SAC, both AFHG extract and roasted garlic prepared at 160 °C (homogenized with 10 mL water/g) were incubated while stirring at pH 8.5 at 37 °C for one hour with a large excess of either protease from Bacillus licheniformis (9.6 units/mg, Sigma-Aldrich) or alkaline protease from Bacillus subtilis (400 PC units/mg, Bio-Cat, Inc., Troy, VA, USA), followed by one hour of incubation with porcine kidney γ-glutamyl transpeptidase (3.4 units/mg, Sigma-Aldrich). Aliquots were centrifuged at 16,000× g and directly assayed for alliin, SAC, and GSAC as described in Section 2.18 .
3
Luria-Bertani and Microbial Assay Protocols
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Luria-Bertani (LB) broth was purchased from Sigma (Sigma-Aldrich, Schnelldorf (Germany)), De Man, Rogosa and Sharpe (MRS) broth and Tryptic Soy (TSB) broth were purchased from Sigma (Millipore, Schnelldorf (Germany)). Folin & Ciocalteu's Phenol Reagent (FC reagent), Triton X-100 (laboratory grade), sodium dodecyl sulfate (SDS; (≥99%)), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; (≥98%)), potassium peroxodisulfate (≥99%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox; (≥97%)), iron(iii ) chloride hexahydrate (FeCl3 × 6H2O; (≥97%)), and protease from Bacillus licheniformis ≥2.4 U were purchased from Sigma (Sigma-Aldrich, Schnelldorf (Germany)). 2,4,6-Tris(2-pyridyl)-S-triazine (TPTZ; (≥98%)) was purchased from Sigma (Supelco, Schnelldorf (Germany)). Coomassie Brilliant Blue R 250 was purchased from Sigma (Sigma-Aldrich, Darmstadt, (Germany)). Peptide digest assay standard and Thermo Scientific PageRuler Plus Prestained Protein Ladder were purchased from Thermo Fisher Scientific (Waltham (USA)). All chemicals used in this research were of analytical grade.
4
Protease-based Protein Hydrolysis
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Protease from Bacillus licheniformis (Alcalase, 2.4 U/g), Aspergillus oryzae (Flavourzyme), bovine serum albumin pH 5.2, ≥96% and trifluoroacetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Corolase 2TS was provided by AB Enzyme (Darmstadt, Germany) and Maxipro AFP, by DSM (Delft, the Netherlands). Millex-HV PVDF 0.45 μm 33 mm filters were used for sample preparation (MilliporeSigma, Burlington, MA, USA).
5
Detecting Glomerular IgA and Gd-IgA1 in Kidney Sections
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Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned at 3 μm for IgA and GdIgA1 staining. Renal slides from MCD, membranous nephropathy, and healthy renal allograft were used as negative controls. Antigen retrieval was performed with 0.05% protease from Bacillus licheniformis (Sigma-Aldrich, St. Louis, MO, United States) at room temperature for 2 h. Then, 3% bovine serum albumin in phosphate-buffered saline was used to block the non-specific binding. Next, the slides were incubated with rat antihuman GdIgA1 antibody (Immuno-Biological Laboratories, Japan) for 1 h at 37°C followed by Alexa Fluor 555-conjugated goat antirat IgG antibody (Abcam, United States) and fluorescein isothiocyanate (FITC)-conjugated polyclonal rabbit antihuman IgA antibody (Dako, Japan) incubation at 37°C for 30 min. Immunofluorescence images were captured using a Nikon microscope 90i (Nikon Instruments Inc., Japan). Glomerular staining of IgA and Gd-IgA1 was evaluated by the Image Pro Plus analysis software version 6.0. Semiquantitative analysis of fluorescence intensity for IgA or GdIgA1 staining was described as the glomerular mean optical density (integrated option density/glomerular area). At least eight fields of glomerular vision per kidney section were randomly captured at 400× for semiquantitative assessment of immunofluorescent staining.
6
Enzymatic Milk Powder Hydrolysis
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Skimmed bovine milk powder (Fonterra & Anchor, Auckland, New Zealand) and hippuryl-histidyl-leucine (HHL) and ACE (Sigma Chemical Co. Ltd., St. Louis, MO) were prepared as the experimental precursors. The commercial proteases used in this study were alcalase (2.4 U/g) (Novozymes, Bagsvaerd, Denmark) and protease from Bacillus licheniformis (2.4 U/g) (Sigma-Aldrich, St. Louis, MO).
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