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Ed at -80 until use and concentrations were determined using a Nanovue PlusTM apparatus (GE Healthcare, Uppsala, Sweden). The study was conducted with the approval of the Research Ethics Committee at the Hospital Infantil Albert Sabin, associated with PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14960617 the Secretary of Health of the State of Cear?Serum preparationFor each sample, high-abundance human serum albumin (HSA) and immunoglobulin G (IgG) proteins were depleted using a HiTrapTM Albumin IgG Depletion column (GE Healthcare) on an TA purifier 10 Fast protein liquid chromatography (FPLC) system (GE Healthcare) according to the manufacturer's instructions. After removal of high-abundance proteins, the flowthrough fractions were collected and stored at -80 until use.Lectin affinity chromatographyThe serum-free high-abundance proteins were thawed, centrifuged for 15 min at 12,000 ?g at 8 , filtered through a 0.22-M membrane (VertipureTM PVDF syringe filters, Veritical) and applied to a 5-mL column packed with Sepharose-Frutalin, prepared as mentioned previously, in a XK16 column on an TA purifier 10 FPLC system (GE Healthcare). The column was washed with five CV of buffer A (20 mM Tris Cl, pH 7.4), and the lectin-bound proteins were eluted with four CV of elution buffer B (20 mM Tris Cl, pH 7.4, with 0.2 M galactose). The eluted protein solution was dialyzed and concentrated by spinning at 8000 ?g (Vivaspin?6, with a molecular weight cut-off of 3 kDa, GE Healthcare), and used for further analyses.Proteomic analysisSerum samples were collected from 3-(2,4-Dichlorophenoxy)azetidine ten pediatric patients with B-ALL at diagnosis and after induction therapy. These patients were diagnosed based on morphological, immunophenotypic, and genetic tests. The study population was composed mainly of children fromBriefly, each sample containing 50 g of protein was denatured with 0.2 RapiGestTM SF (Waters, Milford, USA), reduced with 10 1-(Cyclopropylsulfonyl)-1,4-diazepane mM dithiothreitol, alkylated with 10 mM iodoacetamide, and enzymatically digested withCavalcante et al. Biomarker Research (2016) 4:Page 3 oftrypsin (Promega, Madison, WI, USA). At the end of this process, the samples were centrifuged and the supernatant was transferred oncotarget.13387 to new vials, to which 5 L of internal standard, alcohol dehydrogenase (ADH, 50 fmol, access code P00330 in SwissProt) and 85 L of 3 acetonitrile solution with formic acid 0.1 were added. The final glycoprotein and ADH concentrations were estimated to be 250 ng/L and 25 fmol/L, respectively, in a final volume of 200 L. The quantitative PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/9544797 and qualitative nano-UPLC tandem nano-ESI-MSE experiments were performed on digested samples using peptide reversed-phase chromatography with 3 to 40 (v/v) of acetonitrile containing 0.1 formic acid for 90 min, maintained at a flow rate of 600 nL/min for 100 min in a nanoACQUITY UPLC core system. A nanoACQUITY C18 UPLC BEH 1.7 m, 100 m ?10 cm reversed-phase column was used in conjunction with an SCX 5 m, 180 m ?23 mm pre-column. All analyses were performed with electrospray ionization in positive ion ESI(+) mode and a NanoLockSpray source. Data-independent acquisition (MSE) was performed with a SYNAPT HDMS G1 mass spectrometer (Waters, Manchester, UK), programmed to automatically switch between standard MS (3 eV) and high-energy collision MSE (12?0 eV), applied to trap `T-wave' CID (collision-induced dissociation) cells, in the presence of argon gas. The transfer from the collision cell was adjusted to 1 eV, with 1 s, at both low and high energy. After the time of flight analysis (TOF).