NTCB treatments were performed as previously reported with slight modifications [6 (link)]. Briefly, 8 OD units of cells were treated with 6% trichloroacetic acid (Wako) for at least 15 minutes on ice, washed twice with ice-cold acetone, and dried using a SpeedVac. The pellets were re-dissolved in 200 μl of urea buffer (50 mM Tris-Cl (pH 7.5) (Sigma), 5 mM EDTA (Wako), 6 M urea (Wako), 1% SDS (nacalai tesque), Complete EDTA-free protease inhibitor cocktail (Roche), 1 mM PMSF (Wako), 1 μM microcystin-LR (Wako), and PhosSTOP (Roche)) and lysed with the FastPrep instrument (MP-Biomedicals) and 0.6-mm-diameter zirconia beads (Biomedical Science). After centrifugation at 20,000 × g for 10 min at 4°C, 100 μl of supernatant were transferred to a new 1.5 ml reaction tube. The lysates were mixed with 30 μl of 1 M 2-(cyclohexylamino) ethanesulfonic acid (pH 10.5) (Wako) and 20 μl of 7.5 M NTCB (Sigma) and incubated overnight at room temperature. Each sample was mixed with 50 μl of 4×loading buffer (800 mM Tris-Cl (pH 6.8) (Sigma), 6% SDS (nacalai tesque), 400 mM dithiothreitol (Wako), 8 M urea (Wako), 0.04% bromophenol blue(Sigma)) and subjected to SDS-PAGE and western blot analysis.
Urea
Manufactured by Fujifilm
Sourced in Japan
Urea is a chemical compound that is used in various laboratory equipment. It is a colorless, crystalline solid with the chemical formula CO(NH2)2. Urea is a common ingredient in many laboratory applications due to its ability to act as a denaturant, stabilizer, and buffer.
Automatically generated - may contain errors
Lab products found in correlation
N n n n tetrakis 2 hydroxypropyl ethylenediamine, by Tokyo Chemical Industry (7 mentions)
Urea, by Nacalai Tesque (5 mentions)
Sucrose, by Nacalai Tesque (5 mentions)
Sodium chloride (nacl), by Fujifilm (5 mentions)
Polyethylene glycol mono p isooctylphenyl ether triton x 100, by Nacalai Tesque (4 mentions)
Ethanol, by Fujifilm (3 mentions)
Sucrose, by Fujifilm (3 mentions)
Triton x 100, by Nacalai Tesque (3 mentions)
2 2 2 nitrilotriethanol, by Fujifilm (3 mentions)
Boric acid, by Fujifilm (3 mentions)
2 20 20 nitrilotriethanol, by Fujifilm (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Glycine, by Fujifilm (2 mentions)
Ammonium bicarbonate, by Merck Group (2 mentions)
Disodium hydrogen phosphate, by Fujifilm (2 mentions)
Sulfuric acid, by Fujifilm (2 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (2 mentions)
Formamide, by Fujifilm (2 mentions)
Paraformaldehyde (pfa), by Fujifilm (2 mentions)
Sodium hydroxide (naoh), by Fujifilm (2 mentions)
39 protocols using urea
1
Proteome Profiling Using NTCB Treatment
2
Calcium-depleted bovine αLA characterization
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Calcium-depleted bovine αLA (L-6010) was obtained from Sigma Aldrich (Tokyo, Japan) and used without purification. Urea and DTTred were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). GdmCl was obtained from Tokyo Chemical Industry, Ltd. (Tokyo, japan). DHSox and AEMTS, both of which were not commercially available, were synthesized according to the literature methods [54 (link),55 (link)]. Other common chemicals were procured from local companies and were used as obtained.
3
Immune Response Modulation Protocol
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CHP was purchased from NOF Corporation (Tokyo, Japan). Phosphate-buffered saline (PBS), ATCC-modified RPMI 1640 medium, fetal bovine serum (FBS), 2-mercaptoethanol, and antibiotic-antimycotic were purchased from Gibco (Carlsbad, CA, USA). G418 was purchased from Nacalai Tesque (Kyoto, Japan). EndoGrade OVA was purchased from Hyglos GmbH (Bernried, Germany). InVivoMAb anti-mouse PD-1 antibody (clone J43) was purchased from BioXCell (West Lebanon, NH, USA). Urea was purchased from Wako (Osaka, Japan). Slide-A-Lyzer™ Dialysis Cassettes (10 K MWCO, 3 mL) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). IFN-γ Mouse Uncoated ELISA Kit was purchased from Invitrogen (Carlsbad, CA, USA). CpG with phosphorothioate modification was purchased from FASMAC (Kanagawa, Japan). PE-conjugated anti-mouse CD279 (PD-1) antibody (RMP1-30) and APC-conjugated anti-mouse CD8 antibody (53-6.7) were purchased from BioLegend (San Diego, CA, USA).
4
Fabrication of Polyethersulfone Membranes
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Polyethersulfone (molecular weight of 4800, Sumitomo Chemical Co., Japan) was used as a solute for the membrane. Polyethyleneglycol (PEG, molecular weight of 1000, Wako Pure Chemical Industries, Ltd., Japan) and N,N-dimethylacetamide (DMAc, Wako Pure Chemical Industries, Ltd., Japan) were used as the additive and solvent in the PES casting solution, respectively. An SUS316L plate 200 μm in thickness was purchased from Nilaco Co., Japan. SU-8 3050 (MicroChem Co., Japan) was used as a photoresist for photolithography.
Defibrinated bovine blood used in in vitro experiments was purchased from Kojin Bio Co., Ltd. Sodium chloride, urea, and potassium chloride (KCl) were purchased from Wako Pure Chemical Industries, Ltd., Japan. Saline, heparin, and 32- to 36-week-old Sprague-Dawley rats were used in in vivo experiments and were purchased from Otsuka Pharmaceutical Factory Co., Japan, Mochida Pharmaceutical Co., Ltd., Japan, and Clea Japan, Co., Japan, respectively. Isoflurane purchased from Wako Pure Chemical Industries, Ltd., Japan was used as the anesthesia. Ethanol and 20% glutaraldehyde solution were purchased from Wako Pure Chemical Industries, Ltd., Japan.
Defibrinated bovine blood used in in vitro experiments was purchased from Kojin Bio Co., Ltd. Sodium chloride, urea, and potassium chloride (KCl) were purchased from Wako Pure Chemical Industries, Ltd., Japan. Saline, heparin, and 32- to 36-week-old Sprague-Dawley rats were used in in vivo experiments and were purchased from Otsuka Pharmaceutical Factory Co., Japan, Mochida Pharmaceutical Co., Ltd., Japan, and Clea Japan, Co., Japan, respectively. Isoflurane purchased from Wako Pure Chemical Industries, Ltd., Japan was used as the anesthesia. Ethanol and 20% glutaraldehyde solution were purchased from Wako Pure Chemical Industries, Ltd., Japan.
5
Preparation of Cationic Polymer Complexes
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Once PEI was dissolved in distilled water, the pH of the solution was adjusted with 8 M hydrochloric acid (Nacalai Tesque Inc., Kyoto, Japan, 18321-05) on ice to prevent colorization. Then, urea (Wako Pure Chemical Industries, 210-01185) was dissolved in the pH-adjusted PEI solution at approximately 60 °C using a hot stirrer. Finally, pH of the solution was finely adjusted at 30 °C. Typical final concentrations of PEI and urea were 20 w/v% and 8 M, respectively. Seebest-PP (Propylene oxide-denatured PEI) was prepared in a similar fashion except for the use of propylene oxide-denatured PEI instead of PEI. 4′,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich Co. LLC., Saint Louis, MO, USA) was optionally added to the Seebest solution at a concentration of 5 µg/mL. It was necessary to use freshly prepared Seebest due to the stability and urea crystallization.
6
Amino Acid Quantification Protocol
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All of the amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, ornithine, and citrulline), pyroglutamic acid, urocanic acid, urea, and lactic acid were purchased from Wako (Osaka, Japan). All of the SIL-IS were purchased from JUNSEI (Tokyo, Japan). Deionized water was produced in-house using a Milli-Q gradient water purification system (Merck Millipore, Darmstadt, Germany). The SIL-IS mixture solution containing all of the SIL compounds was prepared in deionized water with each having a final concentration of 0.5 μmol/mL. The NMF mixture solutions containing all of the amino acids and pyroglutamic acid, urocanic acid, urea, lactic acid were prepared in deionized water with a final concentration ranging between 0.025–12.5 μmol/mL for each compound.
7
CUBIC Tissue Clearing Reagent Preparation
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CUBIC reagents were prepared as described15 (link). CUBIC-1 reagent was prepared as a mixture of 25% weight/weight (w/w) urea (Nacalai Tesque, 35904-45, Japan), 25% weight/volume (w/v) N, N, N′, N′-tetrakis (2-hydroxypropyl) ethylenediamine (Tokyo Chemical Industry, T0781, Japan), and 15% (w/v) polyethylene glycol mono-pisooctylphenyl ether (Triton X-100) (Nacalai Tesque, 25987-85, Japan). CUBIC-2 reagent was prepared as a mixture of 50% (w/v) sucrose (Nacalai Tesque, 30403-55, Japan), 25% (w/v) urea, 10% (w/v) 2, 20, 20′-nitrilotriethanol (Wako, 145-05605, Japan), and 0.1% volume/volume (v/v) Triton X-100. Both reagents were prepared just prior to use. Before adding Triton X-100, all other chemicals were dissolved with a hot stirrer at 60 °C. Distilled water was added during a mixing step in order to compensate for water evaporation. After all chemicals except Triton X-100 were dissolved, the solution was cooled to room temperature, and finally Triton X-100 was added.
8
Bacterial Lysis and Sample Preparation
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Zirconium butoxide solution (80%) was purchased from Aldrich (Steinheim, Germany). Iron (II) chloride tetrahydrate was purchased from Acros (New Jersey, USA). Ammonia solution and trifluoroacetic acid were purchased from Reidel-de Haën (Seelze, Germany). Hydrochloric acid and methanol were obtained from J. T. Baker (Phillipsburg, NJ, USA). Nitric acid was purchased from Shimakyu’s Pure Chemicals (Osaka, Japan). Ammonium bicarbonate and iron (III) chloride hexahydrate were obtained from Sigma Chemical Co. (St. Louis, MO). LC–MS grade formic acid and tetraethyl orthosilicate (TEOS) were obtained from Fluka (Buchs, Switzerland). LC–MS grade acetonitrile and coomassie blue G-250 were obtained from Fluka (Buchs, Switzerland). Porcine trypsin (sequence grade) was obtained from Promega (Madison, WI). Urea was obtained from Wako Pure Chemical Industries (Osaka, Japan). Water was purified using a Milli-Q system from Millipore (Bedford, MA). Apple juice, lettuce, and coffee filtering paper were obtained locally in Hualien, Taiwan. All samples were stored at 4 °C before use. The bacteria used in this study, including S. aureus (ATCC 25923), E. coli (ATCC 25922), were cultivated in a BSL-2 safety laboratory at Tzu Chi University. The bacteria were harvested, washed three times with water, boiled in water for 10 min, lyophilized to dryness, and then stored at −20 °C.
9
Synthesis of Nitrogen-Doped Carbon Materials
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All reagents used in these experiments were of analytical grade and were used without further purification. Urea, KOH, and oxalic acid were obtained from FUJIFILM Wako Pure Chemicals. CN was prepared by directly heating Urea to 550 °C in a muffle furnace for 5 h in a covered alumina crucible at a rate of 3 °C/min. Bt-OA-CN was synthesized as follows: 15 g of Urea was dissolved in 30 mL of an aqueous solution of KOH (0.1 g). After stirring for 3 h, the resulting solution was evaporated to dryness at 80 °C using a hot stirrer. The obtained solid was then ground with 5 g of oxalic acid in a mortar. The resultant powder was calcined at 550 °C in a muffle furnace for 5 h at a rate of 3 °C/min. Bt-CN and OA-CN were prepared using the same procedure without the addition of oxalic acid and KOH, respectively.
10
Electrochemical Biosensor for Lactic Acid
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L-lactic acid (CAS: 79-3344, 129-02666), sodium chloride (CAS: 7647-14-5, 195-15975), potassium dihydrogen phosphate (CAS: 7778-77-0,164-22635), disodium hydrogen phosphate (CAS: 7558-79-4, 042-30055), urea (CAS: 57-13-6, 215-00172), ethanol (CAS: 64-17-5, 057-00456), D-glucose (CAS: 50-99-7, 049-31165), L-valine (CAS: 72-18-4, 228-00082), L-leucine (CAS: 61-90-5, 124-00852), and ammonia (CAS: 1336-21-6, 013-17505) were all purchased from Wako, Japan. Lactate oxidase (LOD) from Aerococcus viridans (T-47, Asahi Kasei Pharma, Tokyo, Japan) was purchased from Asahi Kasei Pharma and was used to modify the electrode of the biosensor. Osmium-wired horseradish peroxidase (002096, Bioanalytical Systems, West Lafayette, IN, USA) was purchased from BAS Inc., Tokyo, Japan. Phosphate-buffered saline (PBS, pH 7.4, 50 mM (PO4)) was prepared in the laboratory. Ultrapure water obtained from the water purification system (Direct-Q, Merck, Darmstadt, Germany) was used for the preparation of all aqueous solutions.
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