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发布于:2018-12-21 16:00:51  访问:56 次 回复:0 篇
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Assembled on ice, layering cold solutions of sucrose using a syringe
Assembled on ice, layering cold solutions of sucrose utilizing a syringe with lengthy needle within the following order: 1.5 ml of 1.ten Hygromycin B biological activity density option, 1 ml of 1.13 density answer, 1 ml of 1.15 density remedy, 2 ml of 1.17 density resolution and 2 ml of 1.19 density option. 1A) constituted five distinctive membrane fractions (F1 to F5). The 5 fractions were collected on ice and diluted PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/20954872 to 6 ml with cold 0.1 M Na2CO3, pH 11 and centrifuged in a Type 50 Ti rotor at 100,0006g for 30 min at 4uC. The membrane pellets have been then resuspended in 10 mM Tris-HCl,Materials and Approaches ChemicalsCiprofloxacin (98 purity) was kindly provided as standard for microbiological evaluation by Bayer HealthCare A.G., Leverkusen, Germany. Camptothecin, tunicamycin and also the zwitterionic detergents ASB14 (3-[N,N-Dimethyl(3-myristoylaminopropyl) ammonio]EHop-016 References propanesulfonate Amidosulfobetaine-14) and C7BzO (3-(4Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate) were obtained from Sigma-Aldrich (St. Louis, MO).Cell CultureJ774 mouse macrophages (referred to as WT cells) were grown in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with ten fetal bovine serum in 5 CO2 atmosphere, as described previously [80]. J774 macrophages resistant to ciprofloxacin (known as CIP-R cells) had been obtained as described earlier [4]. In brief, wild-type cells were cultivated within the presence of growing concentrations of ciprofloxacin (from 0.1 mM to 0.2 mM) for about 50 passages (approx. 8 months), soon after which cells were maintained in the presence of 0.two mM ciprofloxacin. MDCKII-MRP1 cells [81] have been received from Prof. P. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25580973 Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands), andCiprofloxacin-Resistant Macrophages and SILACpH 7.four plus protease-inhibitor cocktail-EDTA absolutely free and aliquots have been removed for protein content determination applying the bicinchoninic acid protein assay (Pierce, Rockford, IL). Membrane samples had been then flash-frozen in an ethanol-dry ice bath and stored at ?0uC for downstream evaluation.Western BlotAfter heating for 10 min at 70uC, membrane protein samples had been loaded on acrylamide gels (NuPAGE Bis-Tris 4?2 gels, Invitrogen). After migration, separated proteins had been transferred onto nitrocellulose membranes and after that blocked overnight at 4uC in TBS-T (20 mM Tris-HCl, 500 mM NaCl pH 7.five and 0.05 Tween 20) with five milk. Blotted membranes have been exposed to acceptable principal antibodies, namely rabbit polyclonal antiTLR7 (IMG-581A; Imgenex, San Diego, CA), rat monoclonal antibody anti-MRP1 (MRPr1, Alexis Biochemicals, Lausen, Switzerland), and sc-28259 (Santa Cruz Biotechnology, Santa Cruz, CA) to detect prohibitin, or to antisera against mouse DnajC3 [83], against the amyloid precursor protein APP (A8717, Sigma), or maybe a rabbit antiserum against GRP78 (PA1-014A; Enzo Life Sciences International, Inc., Plymouth Meeting, PA; kindly provided by D. Tyteca, Institute de Duve and Universite ?catholique de Louvain, Brussels, Belgium), followed by suitable horseradish peroxidase-coupled secondary antibody (see figure captions for dilutions). Blots have been revealed by a chemiluminescence assay (SuperSignal West Pico, Pierce).Zorbax 300SB-C18 trapping column, five mm60.three mm, at a 4 ml/ min flow rate employing a two (v/v) acetonitrile, 0.1 formic acid in water buffer.Assembled on ice, layering cold options of sucrose applying a syringe with long needle within the following order: 1.5 ml of 1.10 density answer, 1 ml of 1.13 density answer, 1 ml of 1.15 density resolution, two ml of 1.17 density remedy and 2 ml of 1.19 density resolution.
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