The principal components of bacterial culture: LB broth, tryptone, and yeast extract; antibiotics: kanamycin; protein expression and extraction reagents: IPTG, Tris–HCL and base, glycine, lysozyme, PMSF, SDS, acrylamide/bis-acrylamide, ammonium persulfate, tetramethylethylenediamine (TEMED), DTT, and EDTA, and Coomassie G-250 were purchased from Himedia and VWR life science. Inclusion body solubilizing and protein refolding reagents: guanidine hydrochloride, urea, arginine hydrochloride, and isopropanol were from Sigma-Aldrich. PCR cloning kit: pJET1.2; Q5 high fidelity DNA polymerase and restriction enzymes, T4-DNA ligase, and rapid protein assay BCA kit were from New England Biolabs (NEB) and Thermo Scientific. Protein purification was performed by AKTA FPLC systems using superose 12 10/300 size-exclusion column which were purchased from GE Healthcare. Surface Plasmon Resonance instrument (Autolab ESPRIT) and BLI systems (Octet RED96) were used to measure the binding affinity.
Lysozyme
Manufactured by HiMedia
Sourced in India
Lysozyme is an enzyme that catalyzes the hydrolysis of the peptidoglycan layer in the cell walls of certain bacteria, particularly Gram-positive bacteria. It is found naturally in various bodily fluids, such as tears, saliva, and egg white.
Automatically generated - may contain errors
Lab products found in correlation
Ethylene diamine tetraacetic acid (edta), by HiMedia (4 mentions)
Sodium dodecyl sulfate (sds), by HiMedia (3 mentions)
Imidazole, by HiMedia (3 mentions)
Urea, by Avantor (2 mentions)
Ammonium sulfate, by HiMedia (2 mentions)
Q sepharose, by GE Healthcare (2 mentions)
Hepes, by Merck Group (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Glycerol, by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Potassium hydroxide, by Merck Group (2 mentions)
Potassium chloride (kcl), by HiMedia (2 mentions)
Bovine serum albumin (bsa), by HiMedia (2 mentions)
Ammonium persulfate, by HiMedia (1 mentions)
Arginine hydrochloride, by Merck Group (1 mentions)
Guanidine hydrochloride, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by HiMedia (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by HiMedia (1 mentions)
9 protocols using lysozyme
1
Bacterial Protein Expression and Purification
2
Optimized Protein Purification Procedure
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Bovine serum albumin and lysozyme were purchased from HiMedia Laboratories Pvt. Ltd (India). Calcium chloride (CaCl2), ammonium phosphate dibasic ((NH4)2HPO4), sodium hydroxide (NaOH), anhydrous sodium carbonate (Na2CO3), and ammonia solution were obtained from Rankem Avantor Performance Materials India Limited (Maharastra, India). Sodium phosphate monobasic (NaH2PO4) and sodium phosphate dibasic (Na2HPO4) were obtained from Merck Life Science Private Limited (Mumbai, India). All chemicals were of analytical grade.
3
Oligomer and Fibril Amyloid Assay
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Sodium phosphate dibasic dihydrate, magnesium chloride hexahydrate, ThT, adenosine-5′-triphosphate disodium salt hydrate (ATP), dimethyl sulfoxide, guanidinium hydrochloride, β-mercaptoethanol, and Hepes were procured from Sigma. Urea and PK were bought from Amresco. SDS, imidazole, ammonium sulfate, lysozyme, potassium chloride, and EDTA were purchased from HIMEDIA. Sodium hydroxide, potassium hydroxide, glycerol, methanol, nitrocellulose membrane, A11 (anti–amyloid oligomer antibody), OC (anti–amyloid fibril antibody), horseradish peroxidase–conjugated goat anti-rabbit antibody, and sodium chloride were bought from Merck. Antibiotics (ampicillin and chloramphenicol) and IPTG were procured from Gold Biocom. Q-sepharose and nickel–nitrilotriacetic acid columns were bought from GE Healthcare Lifesciences. Fluorescein-5-maleimide was procured from Invitrogen. About 96-well NUNC optical bottom plates were purchased from ThermoFisher Scientific.
4
Optimized DNA Extraction from Skin Scrapings
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One lesional site of SSS per patient was considered for PCR. Slides were treated with xylene for 2min to remove the oil and washed with water. After complete drying, Phosphate-buffered saline(pH 7.4, 1x PBS: 10 mM PO43−, 137 mM NaCl, and 2.7 mM KCl) 20 μl was added to smear, then scraped with surgical blade and collected in a 1.5 ml micro centrifuge tube. Lysozyme (2mg/ml; 4 μl: HIMEDIA, Mumbai, India) was added, mixed gently, centrifuged at 10,000 rpm for 5sec and then incubated at 37°C for 1hr. Then 150μl of lysis buffer (1 mg/ml Proteinase K and 0.05% Tween 20) was added. About 30μl of 10% Sodium dodecyl sulfate (Sigma Aldrich, Switzerland) was also added to the lysate. The tube containing lysate was centrifuged 30 sec at 10,000 rpm, then vortex for 10–15 sec and again centrifuged for 30 sec. And then it was incubated at 60°C for overnight, then temperature was set at 94°C and the reaction was terminated at 94°C for 15 minutes [16 (link),17 ]. Then purification of DNA by extraction with Phenol:Choloroform method was done as per the method described elsewhere [18 ].
5
Polysulfone Ultrafiltration Membrane Fabrication
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Polysulfone (PSF) for making ultrafiltration membrane was supplied from Solvay. Bovine serum albumin (BSA) (69 kDa) and lysozyme (12 kDa) were obtained from HiMedia Laboratories Pvt Ltd., Mumbai, Maharashtra 400086, India, coliform agar and NMethyl-2-pyrrolidone (NMP) was obtained from Merck KGaA, Darmstadt, Germany. Silver (20, 40, and 90–210 nm) nanoparticles, as shown in Figure 1 [31 ,32 ,33 ], were obtained from Nanostructured & Amorphous Materials, Inc., Katy, TX 77494, USA. Pure water was used for the membrane’s fabrication and water flux test.
6
Purification and Characterization of Amyloid Aggregates
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Hepes, magnesium chloride hexahydrate, sodium phosphate dibasic dihydrate, tris (hydroxymethyl) aminomethane (Tris), β-mercaptoethanol, ATP disodium salt hydrate, DTT, and ThT were bought from Sigma. GdmCl, proteinase K, and urea were procured from Amresco. Ammonium sulfate, imidazole, lysozyme, SDS, EDTA, and potassium chloride was bought from HIMEDIA. Potassium hydroxide, sodium chloride, sodium hydroxide, glycerol, A-11 antiamyloid oligomer antibody, methanol, horseradish peroxide (HRP)–conjugated goat anti-rabbit antibody was procured from Merck. IPTG and antibiotics (chloramphenicol and ampicillin) were purchased from Gold Biocom. Enhanced chemiluminescence kit, HRP-conjugated rabbit antimouse antibody, was obtained from Thermo Fisher Scientific. Nickel-nitriloacetic acid (Ni-NTA) column and Q-Sepharose were from GE Healthcare Lifesciences. Phosphoenolpyruvate (PEP) and pyruvate kinase (PK) were procured from Roche Diagnostics.
7
Recombinant zDHFR Protein Expression and Purification
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Recombinant zDHFR protein were expressed and purified by BL21-DE3 Rosetta E. coli cells. Plasmid pET43.1a, which was carrying zDHFR gene obtained from Dr. Tzu-Fun, Taiwan. Isopropyl-d -1-thiogalactopyranoside (IPTG), Dihydrofolic acid (DHF), Nicotinamide Adenine Di-nucleotide Phosphate (NADPH), Sodium citrate (Na3C6H5O7), Tannic acid (C76H52O46), Silver Nitrate (AgNO3), Oxidized (GSSG) and reduced (GSH) form of glutathione, high purity grade Imidazole, Magnesium chloride (MgCl2), Lysozyme, Phenylmethylsulfonyl fluoride (PMSF), Sodium phosphate buffer, pH 7.4 (Sodium dihydrogen Phosphate (NaH2PO4), Disodium Phosphate (Na2HPO4)), Tris Hydrochloride (Tris HCl), Potassium chloride (KCl), 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB), Sodium chloride (NaCl), Dialysis membrane and Coomassie Blue R-250 were purchased from HiMedia, India. The buffer for unfolding experiments contains 20 mM Tris, 25 mM KCl, 1 mM reduced glutathione (GSH), 0.1 mM oxidized glutathione (GSSG), 10% Glycerol, pH 7.4. Refolding of zDHFR was performed in 20 mM Tris, 25 mM KCl, 10% Glycerol, 1 mM oxidized glutathione (GSSG), pH 7.4. Tris KCl buffer (20 mM Tris, 25 mM KCl, pH 7.4). All other reagents were of analytical grade. Double distilled water or Milli-Q (Merck Millipore) was used for experimentation.
8
Immobilized Organophosphorus Pesticide Detection
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Sephadex G-100 was purchased from MP Biomedicals. Nutrient broth, tris-HCL, EDTA, lysozyme, sucrose, and sodium hydroxide were purchased from Himedia. Glutaraldehyde (grade 1.25%) was purchased from Sigma chemicals, and organophosphorus (OP) pesticide (methyl parathion) was purchased from Shri Ram Fertilizers & Chemicals. All other chemicals were of analytical reagent grade unless otherwise stated. Double distilled water (DDW) was used throughout the experiments. Silver wire and PVC (polyvinyl chloride) beaker were bought from local market.
9
Osteogenic Differentiation of Murine MSCs
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Dulbecco's Modified Eagle's medium (DMEM), fetal bovine serum (FBS), antibiotic-antimycotic solution, ascorbic acid, β-glycerophosphate, dexamethasone, bone morphogenetic protein-2 (BMP-2), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), acridine orange (AO), ethidium bromide (EtBr), alizarin red and lysozyme were purchased from Himedia Laboratories, India. Hexamethyldisilazane (HMDS), phalloidin-FITC (fluorescein isothiocyanate labeled phalloidin), and 4′,6-diamidino-2-phenylindole (DAPI) were acquired from Sigma Aldrich, USA. Murine mesenchymal stem cells (C3H10T1/2 cells) were obtained from National Centre for Cell Science, Pune, India. All the reagents were used as received unless specified otherwise.
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