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发布于:2020-9-15 09:47:46  访问:37 次 回复:0 篇
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D with a 1:twenty dilution of FITC-labeled goat antimouse IgG (Santa Cruz
Right after cooling, the proteins were being separated by SDSPAGE on 12 acrylamide separating gel and 5 acrylamide stacking gels (eighty three ?73 ?one.0 mm) in a very Mini-PROTEAN 3 Cell electrophoresis device (Bio-Rad, Usa) at one hundred twenty V for 2.five h [26]. Right after blotting, theThe surface area antigens ended up precipitated using trichloroacetic acid (TCA) and acetone as with the earlier Enasidenib site explained technique with some modifications [27]. Briefly, the sample was suspended in ten TCA in acetone with 20 mM DTT at -20 for 2 h. Soon after centrifugation at fifteen,000 g at 4 for 15 min, the pellet was resuspended in chilly acetone that contains twenty mM DTT and washed 3 times. The ultimate pellet was air-dried. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25738799 The 2-DE was carried out as previously described [15]. In short, the pellet was suspended in rehydration buffer [7 M urea, 2 M thiourea, four CHAPS, sixty five mM DTT, 0.2 IPG buffer (pH three?0) and 0.001 bromophenol blue], made up of 800 g on the protein samples in the complete volume of five hundred l and centrifuged at 12,000 g for 10 min at home temperature to remove the insoluble elements. The supernatant was loaded onto 24 cm pH four? immobilized pH gradient (IPG) strips (Bio-Rad, United states of america) by over-night reswelling and divided by isoelectric concentrating (IEF) making use of a Protean IEF Mobile (Bio-Rad, United states of america). IEF was performed applying a Protean IEF Cell at 20 as follows: S1: 250 V, 30 min; S2: 500 V, thirty min; S3: a thousand V, 1 h; S4: 10 000 V, 5 h; and S5: ten 000 V, 60 000 Vh (applying a limit of fifty A/strip). After IEF, the IPG strips had been equilibrated sequentially, 1st in equilibration buffer (6 M urea, 0.375 M Tris Cl pH eight.eight, two SDS and twenty glycerol) made up of two dithiothreitol, then in equilibration buffer that contains two.5 iodoacetamide. The 2nd dimension was carried out on twelve SDS-PAGE applying a Mini Protean mobile (Bio-Rad, United states of america). Proteins had been divided for 30 min at 16 mA after which you can at 24 mA until eventually the dye front arrived at the underside of the gel at 16 . Just after 2-DE, proteins were being stained with Coomassie blue R-250 for proteomic investigation as formerly explained [26]. The gel was scanned making use of ImageScanner (GE health care, United states of america). Place detection and location matching have been done by using Graphic Learn second Platinum 6.0 (GE healthcare, United states). A few replicates have been operate to the sample. Only all those protein places that were obviously observed in 3 unbiased experiments ended up selected for even more assessment.2-DE gel excision and tryptic digestion2-DE gel electrophoresis protein spots ended up geared up for MALDI-TOF/TOF-MS evaluation according to standardCui et al. Parasites Vectors 2013, 6:355 http://www.parasitesandvectors.com/content/6/1/Page 4 ofprotocols [28]. Thirty-three spots had been excised manually within the Coomassie blue-stained gels.
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