Cells were collected in RIPA buffer (10 mM Tris-HCl (pH 8.0), 1% (w/v) NP40, 0.1% (w/v) sodium deoxycholate (Wako), 0.1% (w/v) SDS (Wako), 0.15 M NaCl (Wako), 1 mM EDTA, 10 mM NaF (Wako), 1.5 mM Na3VO4 (Wako), and cOmplete™ Protease Inhibitor Cocktail (Roche)). Protein concentrations were titered using the BCA protein assay kit (Thermo Fisher Scientific). Collected protein lysate was mixed with SDS-PAGE loading buffer (0.15 M Tris-HCl, 6% (w/v) SDS, 0.003% (w/v) bromophenol blue (Wako), 30% (w/v) glycerol (Wako), and 15% (w/v) β-mercaptoethanol (Wako)) and then boiled at 95 °C for 5 min, followed by SDS-PAGE and immunoblotting. Antibodies used for immunoblotting were as follows: anti-milk (rabbit, 1:1000; Nordic-MUbio, Susteren, the Netherlands), anti-PyMT (rat, 1:500; Santa Cruz), anti-mCherry (rabbit, 1:500; Abcam), anti-histone H3 (rabbit, 1:2500; Cell Signaling Technology, MA, USA), and anti-α-tubulin (mouse, 1:5000; Calbiochem).
Sodium deoxycholate
Manufactured by Fujifilm
Sourced in Japan, United States
Sodium deoxycholate is a chemical compound commonly used in laboratory settings. It is a bile salt that functions as a surfactant, helping to solubilize and emulsify lipids and other hydrophobic molecules. Sodium deoxycholate is often utilized in various biological and biochemical applications, such as cell lysis, protein extraction, and sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Sodium chloride (nacl), by Fujifilm (3 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (3 mentions)
Sodium dodecyl sulfate (sds), by Fujifilm (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Dojindo Laboratories (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Ethanol, by Fujifilm (2 mentions)
Methanol, by Fujifilm (2 mentions)
Chloroform, by Fujifilm (2 mentions)
Urea, by Fujifilm (2 mentions)
Iodoacetamide, by Fujifilm (2 mentions)
Sodium lauroyl sarcosinate, by Fujifilm (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Fujifilm (1 mentions)
Anti mcherry, by Abcam (1 mentions)
Anti histone h3, by Cell Signaling Technology (1 mentions)
Bromophenol blue, by Fujifilm (1 mentions)
Glycerol, by Fujifilm (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Na3vo4, by Fujifilm (1 mentions)
β mercaptoethanol, by Fujifilm (1 mentions)
20 protocols using sodium deoxycholate
1
Protein Extraction and Immunoblotting
2
Dissection and Homogenization of Brain Regions
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Seven coronal sections of the brain at 2 mm thickness were made, and the third section from the rostral side was collected for the following dissection. The coronal section was dissected into two regions, cerebral cortex and striatum (S), for both hemispheres. The cerebral cortex was further divided into the non-ischemic (N) and ischemic (C) regions. Samples of the six regions in the coronal section were defined as: LN, region of the left cerebral cortex corresponding with the non-ischemic region in the right hemisphere; LC, region of the left cerebral cortex corresponding with the ischemic region in the right hemisphere; LS, striatum in left hemisphere; RN, non-ischemic region of the cerebral cortex in right hemisphere; RC, ischemic region of the cerebral cortex in right hemisphere; and RS, striatum in right hemisphere. Dissected sections were homogenized in lysis buffer (1% Triton X-100; MP Biomedicals (Santa Ana, CA, USA), 0.1% sodium deoxycholate; Wako (Osaka, Japan), 1% EDTA; DOJINDO (Kumamoto, Japan), Complete protease inhibitor cocktail; Roche (Basel, Switzerland), PhosStop phosphatase inhibitor cocktail; Roche, in 50 mM Tris-buffered saline) with Ultrasonic Liquid Processor Q 125 (QSONICA) for 20 s at 4 °C. The homogenate was centrifuged at 15,000 × g for 5 min at 4 °C, and the supernatant was collected for immunoblotting.
3
TLR2 Expression in LPS-Stimulated PEFs
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PEFs_SV40 cells were cultured in 10% FBS DMEM and stimulated with or without 0.1 μg/mL L-LPS for 3 h and 72 h. To obtain total protein extract, cells were lysed in solution which containing 50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 1% Triton X-100, 2.5 mg/mL sodium deoxycholate (Wako Pure Chemical Industries, Osaka, Japan), and a protease inhibitor cock-tail (1:200 dilution, Nacalai-tesque). After calibrating the total protein concentration of cell lysate by a DC Protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA), 10 μg proteins was subjected to SDS-PAGE (ATTO Corporation, Tokyo, Japan) and blotted onto polyvinylidene fluoride membrane (Immobilon-P; Millipore Corporation, MA, USA). The membrane was blotted using the polymer immunocomplexes method as described previously (Fukuda et al., 2000 (link)). TLR2 protein expression level was detected with the ECL chemiluminescence Western blot system for immunostaining (GE healthcare, Buckinghamshire, UK). Anti-α-tubulin antibody (dilution 1:1,000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was used as a loading control.
4
Protein Extraction and Western Blot Analysis
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To extract proteins from WT, K4D, and K4DT cells, we lysed cells in a buffer containing 50 mM Tris–HCl (pH 7.4), 0.15 M NaCl, 1% Triton X-100, 2.5 mg/mL sodium deoxycholate (Wako, Osaka, Japan), and a protease inhibitor cocktail (1/200 dilution, Nacalai Tesque). Total cell lysates were separated by SDS-PAGE and then transferred to PVDF membranes (Merck, Darmstadt, Germany). After the membranes were blocked with 1% nonfat dry milk with 0.05% Tween 20, they were probed with mouse anti-human CDK4 (1:2500, #sc-56277; Santa Cruz Biotechnology, Dallas, TX, USA), Cyclin D1 (1:5000, #553; MBL, Nagoya, Japan), and α-tubulin (1:1000, #sc-32293; Santa Cruz Biotechnology) antibodies. HRP-conjugated goat anti-mouse IgG (1:2000, #330; MBL) or HRP-conjugated goat anti-rabbit IgG (1:2000, #458; MBL) was used as a secondary antibody. Immunoreactive signals were detected using an Image Quant LAS-4000 mini system (GE Healthcare, Little Chalfont, UK) with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).
5
Quantitative Bile Acid Analysis Protocol
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Vancomycin hydrochloride, polymyxin B sulfate, lithocholic acid, sodium deoxycholate, sodium taurocholate, and lysyl endopeptidase were purchased from Wako Pure Chemical Industries (Osaka, Japan). QIAamp Fast DNA Stool Mini Kit was from Qiagen (Hilden, Germany). Taq DNA polymerase containing 10 × Standard buffer (Taq) and dNTPs were obtained from BioAcademia (Osaka, Japan) and synthesised PCR primers were obtained from FASMAC (Kanagawa, Japan). Sodium taurolithocholate and sodium taurochenodeoxycholate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Taurodeoxycholic acid sodium salt was purchased from Nacalai Tesque (Kyoto, Japan). Tauro β-muricholic acid sodium salt was purchased from Steraloids (Newport, RI, USA). Taurocholic acid-d5 (TCA-d5) sodium salt was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). Triglyceride Quantification Assay kit was purchased from Abcam (Cambridge, UK). Plasma Membrane Protein Extraction Kit was obtained from BioVision (Milpitas, CA, USA). Pierce BCA Protein Assay Kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Sequencing-grade modified trypsin (frozen) was obtained from Promega (Madison, WI, USA). Synthesised isotope-labelled peptides were obtained from Sigma-Aldrich. Other reagents were commercially available products of reagent or analytical grade.
6
3D Ultrastructural Analysis of Acellular Glomeruli
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To evaluate the 3D ultrastructural findings of GBM surface, acellular glomeruli after acellular treatment, that is, digestion of all cellular components, were prepared as described previously [17 (link), 18 (link)]. The small renal cortex tissues (2–3 mm3) were sequentially exposed to 5 mM EDTA (Sigma Aldrich Corp, St. Louis, MO, USA) for 24 h (h), 3% Triton X-100 (Sigma Aldrich Corp, St. Louis, MO, USA) for 18 h, 0.025% deoxyribonuclease (Worthington Biochemical Crop. Lakewood, USA) in 1 M NaCl for 6 h, and 4% sodium deoxycholate (FUJIFILM Wako Pure Chemical Corp Tokyo, Japan) for 18 h. After t-butyl alcohol vacuum drying, freeze-dried specimens were coated with gold powder and examined by SEM (SU3500, Hitachi, Tokyo, Japan) and LV-SEM (Hitachi Tabletop Microscope TM3030 plus, Hitachi High-Technologies Corp., Tokyo, Japan). Acellular glomeruli stained with uranyl acetate and lead citrate or PAM were also examined by TEM.
7
Phosphoproteome Enrichment and Analysis
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Cells were washed and harvested with ice-cold PBS. The proteins were extracted with phase-transfer surfactant (34 (link)) using a lysis buffer (12 mM sodium deoxycholate [Fujifilm Wako], 12 mM sodium N-lauroylsarcosinate [Fujifilm Wako], 100 mM Tris-HCl [pH 9.0], containing protein phosphatase inhibitor cocktail 1 and 2 [Sigma-Aldrich], and protease inhibitors [Sigma-Aldrich]). Protein amount was determined with a BCA protein assay kit, and the proteins were reduced with 10 mM DTT for 30 min and then alkylated with 50 mM iodoacetamide for 30 min in the dark. After reduction and alkylation, proteins were digested with Lys-C (w/w 1:100) for 3 h, followed by trypsin digestion (w/w 1:100) overnight at 37 °C. Then, the peptides were desalted using SDB-XC StageTip.
Phosphopeptides were enriched from 100 μg of tryptic peptides by means of TiO2-based hydroxy-acid-modified metal oxide chromatography (HAMMOC) (35 (link)) and eluted with 0.5% piperidine. Phosphopeptides were labeled with tandem mass tag (TMT) (Thermo Fisher Scientific), desalted using SDB-XC StageTips, fractionated at basic pH (33 (link)), and suspended in the loading buffer (0.5% TFA and 4% ACN) for subsequent LC/MS/MS analyses.
Phosphopeptides were enriched from 100 μg of tryptic peptides by means of TiO2-based hydroxy-acid-modified metal oxide chromatography (HAMMOC) (35 (link)) and eluted with 0.5% piperidine. Phosphopeptides were labeled with tandem mass tag (TMT) (Thermo Fisher Scientific), desalted using SDB-XC StageTips, fractionated at basic pH (33 (link)), and suspended in the loading buffer (0.5% TFA and 4% ACN) for subsequent LC/MS/MS analyses.
8
Cisplatin-Loaded Nanoparticle Formulation
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Cisplatin was purchased from Wako Pure Chemical Industries Co., Ltd. (Tokyo, Japan). Egg phosphatidylcholine (EPC, Coatsome® NC-50) and Tween 80 were products of NOF Inc. (Tokyo, Japan). Sodium Deoxycholate (SD) was acquired from Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan). Sodium cholate (SC) was supplied by Sigma Aldrich (Tokyo, Japan). The diblock polymer used in this experiment was polyethylene oxide-b-polymethacrylic acid (PEO-b-PMAA; Mw of PEO = 7500; Mn of PMAA= 11 000) was obtained from Polymer Source, Inc. (Canada). Saline was purchased from Otsuka Co. Ltd. (Japan). In order to undertake high-performance liquid chromatography (HPLC) analysis, all solvents were of HPLC analytical grade. For graphite furnace atomic absorption spectrophotometry (GF-AAS) measurements, nitric acid (1.38; analytical grade, Wako Pure Chemical Industries Co., Ltd., Osaka, Japan) was employed as a digestive acid solution, while hydrochloric acid (AAS analytical grade) was used for the sample solvent (Kanto Chemical Co., Inc., Tokyo, Japan). The solvents; ethanol, methanol, acetone, and chloroform; were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents and solvents employed in this study were of the highest quality available. Milli-Q water was used in all experiments.
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Cisplatin was purchased from Wako Pure Chemical Industries Co., Ltd. (Tokyo, Japan). Egg phosphatidylcholine (EPC, Coatsome® NC-50) and Tween 80 were products of NOF Inc. (Tokyo, Japan). Sodium Deoxycholate (SD) was acquired from Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan). Sodium cholate (SC) was supplied by Sigma Aldrich (Tokyo, Japan). The diblock polymer used in this experiment was polyethylene oxide-b-polymethacrylic acid (PEO-b-PMAA; Mw of PEO = 7500; Mn of PMAA= 11 000) was obtained from Polymer Source, Inc. (Canada). Saline was purchased from Otsuka Co. Ltd. (Japan). In order to undertake high-performance liquid chromatography (HPLC) analysis, all solvents were of HPLC analytical grade. For graphite furnace atomic absorption spectrophotometry (GF-AAS) measurements, nitric acid (1.38; analytical grade, Wako Pure Chemical Industries Co., Ltd., Osaka, Japan) was employed as a digestive acid solution, while hydrochloric acid (AAS analytical grade) was used for the sample solvent (Kanto Chemical Co., Inc., Tokyo, Japan). The solvents; ethanol, methanol, acetone, and chloroform; were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents and solvents employed in this study were of the highest quality available. Milli-Q water was used in all experiments.
9
Isolation and Culture of Primary Cells
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Hygromycin B Gold was purchased from InvivoGen Co. (San Diego, CA, USA). HEPES was purchased from Dojindo Laboratories (Kumamoto, Japan). Hank's balanced salt solution was purchased from Sigma-Aldrich (St. Louis, MO, USA). Deoxyribonuclease I from bovine pancreas, precrystalline, DISPASE II, NP-40 substrate, tris (hydroxymethyl) aminomethane, sodium deoxycholate, sodium dodecyl sulfate, chloroform, isopropyl alcohol, ethanol, NaHCO3, d-(+)-glucose, and 0.4 w/v % trypan blue solution were purchased from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). Collagenase D was purchased from Roche Diagnostics, Inc. (Mannheim, Germany). Fetal bovine serum (FBS) was purchased from Biosera (East Sussex, UK). Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan). Mitomycin C, a penicillin-streptomycin-glutamine mixed solution, and 100 mM sodium pyruvate solution (100 × ) were purchased from Nacalai Tesque Inc. (Kyoto, Japan). All the other chemicals used were of the highest commercially available grade.
10
Protein Extraction and Western Blot Analysis
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To extract proteins from primary, K4D, and K4DT + T cells, we lysed cells in a buffer containing 50 mM Tris–HCl (pH 7.4), 0.15 M NaCl, 1% Triton X-100, 2.5 mg/ml sodium deoxycholate (Wako, Osaka, Japan; cat. no. 194-08311), and a protease inhibitor cocktail (1/200 dilution, Nacalai Tesque; cat. no. 25955-11). The protein expression levels of CDK4 and Cyclin D were detected by Western blotting using an anti-CDK4 antibody (1/2,500 dilution, MBL, Nagoya, Japan; cat. no. 25955-11), an anti-cyclin D antibody (1/5,000 dilution, MB; cat. no. 553), and an anti-alpha-tubulin antibody (1/1,000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA; cat. no. sc-32,293). Anti-mouse IgG (1/2,000 dilution, GE Healthcare, Buckinghamshire, UK; cat. no. NA931) or anti-rabbit IgG (1/2,000 dilution; GE Healthcare; cat. no. NA934-1ML) was used as secondary antibodies. The detailed Western blot procedure has been reported previously (Fukuda et al., 2005 (link), 2008 (link)). The intensity of signals was measured by the Image J software.
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