Cultures of strains 168 and 168L were harvested by centrifugation at 16 000 × g for 20 min at 4 °C at the late exponential phase of growth. Pellets were washed three times with 10 mM TRIS–HCl (pH 7.4) and resuspended in protein extract consisting of 1.52 g thiourea, 4.2 g urea, 0.4 g CHAPS, 200 µL amphoteric electrolyte, 61.6 mg dithiothreitol (DTT; all Bio-Rad), and protease inhibitor (Merck) dissolved in 10 mL ultrapure water. After vortexing and centrifugation, total protein in the supernatant was subjected to cleanup with a ReadyPrep 2-D cleanup kit (Bio-Rad). Purified proteins were redissolved in 350 µL rehydration solution (7 M urea, 2 M thiourea, 0.001% bromophenol blue; Bio-Rad) and centrifuged to remove insoluble components. Samples were finally loaded onto a 17 cm strip (pH 3–10; Bio-Rad) and isoelectric focusing (IEF) was carried out at 20 °C by positively rehydrating at 50 V for 12 h, increasing slowly to 250 V for 1 h, rapidly to 1000 V for 1 h, 10 000 V for 3 h, rapidly to 10 000 V to a total of 90 000 Vh, then rapidly to 500 V. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was then performed according to a previous protocol [24 (link)].
Thiourea
Manufactured by Bio-Rad
Sourced in United States
Thiourea is a chemical compound with the formula CS(NH2)2. It is a white crystalline solid that is soluble in water and various organic solvents. Thiourea is commonly used as a reducing agent and as a building block in the synthesis of other organic compounds.
Automatically generated - may contain errors
Lab products found in correlation
Urea, by Bio-Rad (11 mentions)
Dithiothreitol (dtt), by Bio-Rad (8 mentions)
Mineral oil, by Bio-Rad (4 mentions)
Chaps, by Bio-Rad (4 mentions)
Chaps 3 3 cholamidopropyl dimethylammonio 1 propanesulfonate, by Bio-Rad (4 mentions)
Readyprep 2 d cleanup kit, by Bio-Rad (3 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Amphoteric electrolyte, by Bio-Rad (2 mentions)
Iodoacetic acid, by Merck Group (2 mentions)
Sodium dodecyl sulfate (sds), by Bio-Rad (2 mentions)
Bis acrylamide, by Merck Group (2 mentions)
Pierce 660 nm protein assay kit, by Thermo Fisher Scientific (2 mentions)
Proteomics grade trypsin from porcine pancreas, by Merck Group (2 mentions)
Anti age monoclonal antibodies for western blotting, by MP Biomedicals (2 mentions)
C 18 ziptip columns, by Merck Group (2 mentions)
Ficoll histopaque density gradient, by Merck Group (2 mentions)
Ipg strip, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions)
Ammonium bicarbonate, by Merck Group (2 mentions)
12 protocols using thiourea
1
Proteomic Analysis of Bacillus subtilis
2
Protein Purification and Isoelectric Focusing
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The protein mixture obtained by TRIS extraction assisted with glass beads was purified, desalted, and concentrated by Amicon Ultra-0.5 3 kDa centrifugal filter units (Merck KGaA, Darmstadt, Germany). The concentrated solution was then subjected to protein precipitation using a precipitation kit (Ready-Prep™ 2-D Cleanup Kit, Bio-Rad, Warsaw, Poland) to obtain a pellet containing 200 µg of protein. The protein precipitate was dissolved in 300 µL of rehydration buffer containing thiourea (Bio-Rad, Warsaw, Poland), transferred to a rehydration plate, and covered with 17 cm immobilised pH gradient linear strips for isoelectric focusing (pH 3–10, Bio-Rad, Warsaw, Poland) flooded with mineral oil (pH 3–10, Bio-Rad, Warsaw, Poland) and left overnight [12 ].
3
Mammary Tissue Protein Extraction
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Total protein was extracted from mammary tissues using 10% trichloroacetic acid (TCA) (Beyotime, Beijing, China) (prepared with acetone, containing l g of TCA and 0.07% β-mercaptoethanol). And then, 1 mL of the liquid mixture was added to a centrifuge tube, shaken for 5 min, kept overnight, and centrifuged at 12,000 g, 4 °C for 40 min. The supernatant was discarded and washed with cold acetone, and then centrifuged at 12,000 g, 4 °C for 10 min. After the supernatant was discarded, the steps were repeated three times. The pellet was dried and then dissolved in lysis buffer containing 7 M urea (Bio-Rad, Hercules, CA, USA), 2 M thiourea (Bio-Rad, Hercules, CA, USA), 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (Bio-Rad, Hercules, CA, USA), 40 mM dithiothreitol (DTT) (Solarbio, Beijing, China), 40 mM Tris (Solarbio, Beijing, China)and 2% immobilized pH gradients (IPG) buffer (ReadyStripTM IPG strips, Bio-Rad, Hercules, CA, USA). After centrifugation at 1000 g, 4 °C for 10 min, the supernatant was collected, quantified, and then stored at −80 °C for 2-DE analysis.
4
Nail Keratins Extraction from Diabetic and Non-Diabetic Individuals
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The extraction of nail keratins was carried out by the Shindai method [33 (link)]. Nail clippings from diabetic and nondiabetic individuals were collected and washed with ethanol before delipidization into the mixture chloroform/methanol (2:1, V/V, Merck, Darmstadt, Germany) for 24h. The extraction of nails was done using a solution containing 25 mmol/L Tris-HCl, pH: 8.5, 2.6 mol/L thiourea, 5 mol/L urea (Bio-Rad Laboratories Inc., Richmond, CA, USA) and 5% 2-mercaptoethanol (2-ME, Fluka, St. Louis, MO, USA) at 50°C for 3 days. The mixture was filtered and centrifuged at 15,000 g (20 min, room temperature). The obtained supernatant was used as nail protein extract.
5
Comprehensive Proteomics Sample Preparation
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All reagents used were of molecular grade, protease inhibitor cocktail, Phenylmethylsulfonyl fluoride (PMSF), Proteomics grade trypsin from porcine pancreas, Iodoacetic acid (IAA), Acrylamide, bis Acrylamide, ficoll histopaque density gradient and Ammonium bicarbonate were procured from Sigma-Aldrich, St. Louis, MO, USA. Sodium Dodecyl Sulfate (SDS), IPG strips, Urea, thioUrea, Mineral oil, Dithiothreitol (DTT), CHAPS (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) were purchased from Bio-Rad Laboratories, Inc. California, USA. For protein quantification, Pierce™ 660nm Protein Assay kit from Thermo Fisher Scientific Inc. USA was used. Mouse, Anti-AGE monoclonal antibodies for western blotting were obtained from MP Biomedicals, USA. The polyvinylidenedifluoride (PVDF) membrane (0.45μm) and dialysis tubing were purchased from Millipore Corporation, MA, USA. C-18 ZipTip columns for concentrating and desalting the peptides were purchased from Millipore, Billerica, MA, USA. Pre-stained protein ladder was procured from Real Biotech Corporation, Taipei, Taiwan.
6
Two-Dimensional Gel Electrophoresis Reagents
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Immobilized pH gradient (IPG) strips (pH 3.0 to 10.0 non-linear (NL); 17 cm), urea, thiourea, dithiothreitol (DTT), 3-([3-cholamidopropyl] dimethyl-ammonio)-1-propane sulfonate (CHAPS), ampharmalyte pH 3 to 10, phenylmethane sulfonyl fluoride (PMSF), iodoacetamide (IAM) were purchased from Bio-Rad (Richmond, CA, USA). Coomassie brilliant blue G-250 was purchased from AMRESCO (Solon, OH, USA). All chemicals for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were of electrophoresis grade.
7
Extraction of Membrane Proteins from C. sakazakii
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Two C. sakazakii isolates (G362 and L3101) were inoculated into tryptic soy broth (TSB, Huankai, Guangzhou) for incubation at 37°C for 16 h. Two grams (wet weight) of G362 and L3101 were suspended in 5 ml of phosphate buffer saline (PBS, pH7.4) at 4°C. Then the cell suspension was sonicated in an ice bath (SONICS VC-505). Following the sonication process, Triton X-114 was applied for membranous proteins extraction using the method described by González de la Vara and Alfaro (2009) (link). Then, two different phase collections and the insoluble pellet were performed to analyze SOD activity (Huang et al., 2006 (link)). Acetone precipitation was employed to remove the excess salts in the aqueous phase collection and detergent phase collection respectively. After lysis buffer (5 M urea, 2 M thiourea, 2% SB3-10, 2% CHAPS, 65 mM DTT, 40 mM Tris; Bio-Rad, USA) dissolution and centrifuged at 18, 000 ×g for 1 h at 4°C, membrane proteins supernatant was stored at -80°C for use. Proteins qualification was measured by the bicinchoninic acid (BCA) assay as described by Krieg et al. (2005) (link).
8
Protein Extraction and Quantification Protocol
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The obtained culture was centrifuged at 12,000 rpm for 20 min at 4°C. The precipitates were washed three times with sterile 10 mM Tris-HCl (pH 7.4) and resuspended in 30 μL protein extract (1.52 g thiourea [Bio-Rad], 4.2 g urea [Bio-Rad], 0.4 g CHAPS [Bio-Rad], 200 μL amphoteric electrolyte [Bio-Rad], 61.6 mg DTT [Bio-Rad], and protease inhibitor [Merck] dissolved in 10 mL ultrapure water). After vortexing for 15 s, the mixture was placed in an ice bath for 30 s, and the procedure was repeated for 15 min. The whole protein in the supernatant was obtained by centrifugation at 12,000 rpm for 30 min at 4°C. A ReadyPrep 2-D cleanup kit (Bio-Rad, USA) was used to clean up the protein samples. The protein concentration was determined using a BCATM Protein Assay Kit (Themo Scientific, USA).
9
Rat Model of Alzheimer's Disease
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Fibrauretine (Yunnan Plant Pharmaceutical Co., Ltd., State Drug Quotient: Z53020154, Lot No.: 1407067), Protein lysis buffer (7M urea, 2M thiourea, 0.1% CHAPS), urea (Bio-Rad, Lot No.: 161-0731, USA), Thiourea (Sigma-Aldrich, Lot No.: T7875, USA), CHAPS (Bio-Rad, Lot No.: 161-0460, USA), Protease inhibitor (Shanghai Limin Industrial Co., Ltd., model: 04693132001/kit), Ultrasonic cell disruptor (Nanjing Xianou Instrument Manufacturing Co., Ltd., model: XO), Orbitrap Fusion (Thermo, model: Orbitrap Fusion), Aβ 1-40 (MedChemExpress, Lot No.: 131438-79-4), sodium carboxymethylcellulose (Shanghai Yuanye Biotechnology Co., Ltd., Lot No.: 9004-32-4), SD rats (male, weight 190-220 g, SPF grade). All rats were provided by YISI Experimental Animals Co., Ltd. (license No.SCXK (Ji)-2020-0002). All animals were acclimatized and fed at a humidity of 60% ± 10% and room temperature of 23°C ± 2°C for 1 week, and all rats were allowed to drink and eat freely during the experimental period [7] (link). The Animal Care and Use Committee of the Academy of Chinese Medical Sciences approved these animal studies (Animal Ethics No: 2021 07 26001), which were then carried out in accordance with the "Guide for the Care and Use of Laboratory Animals" [8] (link).
10
Mitochondria Isolation and Enzyme Assay
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Isoelectric pH gradient (IPG) strips (pH 3.0–10.0; NL, 17 cm), urea, pharmalyte (pH 3–10), glycerol (87% w/w), Tris (electrophoresis grade), 1,2-di(dimethylamino)ethane (TEMED; electrophoresis purity reagent), acrylamide (40% solution; acrylamide-to-bisacrylamide ratio, 37.5:1), 3-[(3-cholamidoprpyl) dimethylammonio]-1-propanesulfonate (CHAPS; electrophoresis grade), thiourea (ACS grade), dithiothreitol (DTT, electrophoresis grade), iodoacetamide (electrophoresis grade), mineral oil, Coomassie G-250 stain and low-melting-point agarose were obtained from Bio-Rad. Animal cell/tissue quality purified mitochondria isolation kits were purchased from Genmed Scientifics, and the enzyme activity assay kits were purchased from Nanjing Jiancheng Biotechnology Institution. High-purity water prepared from the Milli-Q gradient water purification system (Millipore) was used for all the experiments in this study.
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