Sodium chloride, glycerol, sodium phosphate saline (PBS), isopropyl β-d-1-thiogalactopyranoside (IPTG), Tris HCL and base, Glycine, lysozyme, dithiothreitol (DTT), and ethylene-di-aminetetraacetic acid (EDTA), Coomassie G-250 were purchased from VWR life science. Inclusion body (IB) solubilizing and protein refolding reagents: GSH, GSSG, L-Arginine, and urea were from Sigma-Aldrich (St. Louis, MI, USA)). PCR cloning kit: PET series vectors; all restriction enzymes; T4 DNA ligase and rapid protein assay BCA kit were from New England Biolabs (NEB, Ipswich, MA, USA) and Thermo Scientific (Cincinnati, OH, USA). ELISA reagents: Human serum albumin (HAS), bovine serum albumin (BSA), Tween-20, Citric acid, H2O2, o-phenylenediamine dihydrochloride (OPD) were from Sigma-Aldrich and Alfa Aesar. Protein purification apparatus and affinity chromatography columns were purchased from GE Healthcare.
Lysozyme
Manufactured by Avantor
Sourced in United States
Lysozyme is an enzyme that catalyzes the hydrolysis of the peptidoglycan layer in bacterial cell walls. It is a naturally occurring substance found in various biological sources, including egg whites, tears, and certain other bodily fluids.
Automatically generated - may contain errors
Lab products found in correlation
Acetonitrile, by Thermo Fisher Scientific (3 mentions)
Ni nta agarose, by Qiagen (2 mentions)
Mutanolysin, by Merck Group (2 mentions)
Lysostaphin, by Merck Group (2 mentions)
Proteinase k, by Research Products International (2 mentions)
Zr fecal dna miniprep kit, by Zymo Research (2 mentions)
Fastprep 24, by MP Biomedicals (2 mentions)
Glycine, by Avantor (1 mentions)
Dithiothreitol (dtt), by Avantor (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
O phenylenediamine dihydrochloride, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Avantor (1 mentions)
Rapid protein assay bca kit, by New England Biolabs (1 mentions)
Tween 20, by Merck Group (1 mentions)
L arginine, by Merck Group (1 mentions)
Citric acid, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Urea, by Avantor (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
18 protocols using lysozyme
1
Recombinant Protein Expression and Purification
2
Novel Membrane Fabrication for Filtration
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Utilizing the procedure from Moore et al., OTO-CNMs Form I and Form II were synthesized and used in conjunction with cellulose triacetate (CTA) from Acros Organics (Fair Lawn, NJ, USA), N-methyl pyrrolidone (NMP) from VWR (Radnor, PA, USA), and deionized water to create novel membranes for filtration [21 (link)]. The proteins used for ultrafiltration and dialysis characterization were bovine serum albumin (BSA), lysozyme, and urea supplied from VWR. These are common proteins found in the blood. BSA was chosen to represent large proteins in the blood; lysozyme was selected to represent middle molecular weight proteins in the blood, and urea was chosen because it is the primary waste product that is excreted from the kidneys. All membranes were housed in polyvinyl chloride pipes (PVC) supplied from Lowe’s (Fayetteville, AR, USA) using underwater epoxy resin. Ethanol for cleaning was also provided through VWR.
3
Salmonella Typhimurium Cell Lysis Protocol
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One-liter cultures were collected at specific growth phases and centrifuged at 8,000 rpm. The pellets were resuspended in 10 mL of PBS and incubated for 30 min at 4°C with lysozyme (144 µg/mL) (VWR Cat. # 97062-136), 1 µL of protease inhibitor cocktail EDTA-free (100×) (Thermo Cat. # 78425), and 1 µL of RNAse/DNAse nuclease reagent (Sigma Cat. # 70746-3). The treated S. Typhimurium bacteria were homogenized and lysed using Emulsiflex-C3 (Avestin) until the sample was clear. After centrifugation at 8,000 rpm, the whole-cell lysates were ready to use or freeze. If total membrane harvesting was desired, the previous lysate was ultracentrifuged at 40,000 rpm for at least 2 h at 4°C. Membranes were resuspended in 1 mL of PBS.
4
Bisabolol Compound Purification and Analysis
5
C. elegans Longevity Assay with EGCG
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The N2 wild-type Caenorhabditis elegans (C. elegans) strain and E. coli OP50 were purchased from Caenorhabditis genetics Center (University of Minnesota, Minneapolis, MN, USA). Lysozyme (enzyme activity > 20 ku/mg) was obtained from VWR International, Inc. (St. Louis, MO, USA). Pectin (galacturonic acid (dry basis) ≥ 74.0%) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Epigallocatechin-3-gallate (EGCG) (98% purity) was provided from Chengdu purefa Technology Development Co., Ltd. (Chengdu, China).
6
Mycotoxin Detection and Enzyme Purification
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AFB1, ZEN, DON, and the commercial Nematoloma frowardii manganese peroxidase (NfMnP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). FB1 and OTA were purchased from Pribolab (Beijing, China). Hemin was purchased from TCI (Tokyo, Japan). DNA polymerase, T4 ligase, and chromatographic grade reagents (acetonitrile, methanol, methanoic acid, acetic acid, and trifluoroacetic acid) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). DNase I and TransScript One-Step gDNA Removal and cDNA Synthesis Supermix with oligo(dT) were purchased from TransGen (Beijing, China). Isopropyl-β-D-thiogalactoside (IPTG), SOD, DTT, and Rutin were purchased from Solarbio (Beijing, China). TRIZOL was from Invitrogen (Carlsbad, CA, USA). Lysozyme was purchased from Amresco (Solon, OH, USA). Ni-NTA agarose was purchased from QIAGEN (Duesseldorf, Germany). All other chemicals were of analytical grade or chromatographically pure, and were commercially available.
7
Purification of PKCβ C2 Protein Domains
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N-terminal GST fusion proteins of PKCβ C2WT and C2D/A were expressed in Escherichia coli BL21 cells. Pelleted bacteria were resuspended in ice-cold PBS supplemented with 500 µM EDTA, 0.5 mg/ml lysozyme (Amresco, Solon OH), and protease inhibitor cocktail (Easypack; Roche, South San Francisco CA), and the bacteria were lysed by sonication. After centrifugation at 11,200 RPM for 30 min, the soluble fraction was collected and incubated with glutathione sepharose 4B beads (GE healthcare, Pittsburgh PA) for 1 hr at 4°C. Samples were cleared from nucleic acid contaminants with benzonase (40 U/ml, Sigma) for 3 hr at RT, and subsequently eluted from the beads with solution containing 100 mM Tris, 10 mM CaCl2, 5 mM Glutathione (pH 7.4) for 1 hr at 4°C. GST was cleaved with thrombin-agarose (100 µl resin/mg protein, Sigma) for 24 hr at 4°C, and samples were dialyzed to solution containing 40 mM Tris–HCl pH 7.4, 100 mM NaCl, and 0.5 mM Na-EGTA. GST was removed from the samples using glutathione sepharose 4B beads. 10 µl of purified protein was run on a 12% SDS gel and Coomassie blue-stained to check for purity (Figure 2B ).
8
DNA Extraction from Stool Samples
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DNA was extracted from each of 283 stool specimens. For processing, samples were thawed at 4°C and, in aliquots of 0.15 g per tube, resuspended in 1 ml of 1× phosphate-buffered saline. Cell lysis was initiated with two enzymatic incubations, first, using 5 µl of lysozyme (10 mg ml−1; Amresco, Solon, OH), 13 µl of mutanolysin (11.7 U µl−1; Sigma-Aldrich), and 3 µl of lysostaphin (4.5 U µl−1; Sigma-Aldrich) for an incubation of 30 min at 37°C and, second, using 10 µl proteinase K (20 mg ml−1; Research Products International, Mt. Prospect, IL), 50 µl 10% SDS, and 2 µl RNase (10 mg ml−1) for an incubation of 45 min at 56°C. After the enzyme treatments, cells were disrupted by bead beating in tubes with lysing matrix B (0.1-mm silica spheres; MP Biomedicals, Solon, OH), at 6 m s−1 for 40 s at room temperature in a FastPrep-24 (MP Biomedicals). The resulting crude lysate was processed using the ZR fecal DNA miniprep kit (Zymo, Irvine, CA) according to the manufacturer’s recommendations. The samples were eluted with 100 µl of ultrapure water into separate tubes. DNA concentrations in the samples were measured using the Quant-iT PicoGreen double-stranded DNA (dsDNA) assay kit (Molecular Probes, Invitrogen, Carlsbad, CA).
9
Peptide Synthesis and Characterization
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Fmoc-protected amino acids and Wang resin were purchased from Novabiochem®. OymaPure®, N-ethyldiisopropylamine, piperidine and trifluoroacetic acid were obtained from Carl Roth. Dimethylformamide (DMF for peptide synthesis), diethyl ether and dimethylsulfoxid was purchased from Acros Organics. Acetonitrile was purchased from Fisher Scientific. Uranyl acetate and fluorescamine were purchased from Merck. Lysozyme was purchased from Amresco. PBS, Thioflavin T and α-cyano-4-hydroxycinnamic acid were purchased from Sigma Aldrich. Proteostat® was purchased from Enzo Life Sciences. All chemicals are listed in Supplementary Data 1 and were used as received unless explicitly stated otherwise.
10
Immobilized Enzymes for Antimicrobial Screening
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β-Galactosidase from Aspergillus oryzae (with activity not less than 14,000 U/g) was purchased from Amano Enzymes Co. (Nagoya, Japan). Tetraethylorthosilicate (TEOS) and Pluronic P123 (EO20PO70EO20, Mav = 5800) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-Aminopropyltriethoxysilane (APTES) was purchased from Fluka (Buchs, Switzerland). Lysozyme was purchased from Amresco (Solon, OH, USA). Spectral-grade KBr was purchased from BDH Co., Ltd. (Poole, UK). The glucose oxidase-peroxidase kit was purchased from Beijing BHKT Clinical Reagent Co., Ltd. (Beijing, China). All other chemicals and reagents were of analytical grade. All aqueous solutions were prepared with Milli-Q water (Millipore, Billerica, MA, USA). Micrococcus lysodeikticus ATCC No. 4698 cells were obtained from Sigma-Aldrich. Bacterial strains Staphylococcus aureus ATCC 653 and Escherichia coli ATCC 1122 were purchased from the China General Microbiological Culture Collection Center (CGMCC, Beijing, China).
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