Adenovirus: Methods and Protocols by Rosa Menéndez-Conejero, Ana J. Pérez-Berná, Gabriela N.

By Rosa Menéndez-Conejero, Ana J. Pérez-Berná, Gabriela N. Condezo (auth.), Miguel Chillón, Assumpció Bosch (eds.)

Reflecting the advance of robust new instruments and high-throughput ways to research adenoviral debris and their interactions with host cells, the 3rd variation of Adenovirus tools and Protocols calls upon specialists within the box to show advances in molecular biology, genomics and proteomics, imaging, and bioinformatics. starting with cryo-electron microscopy, atomic strength microscopy, and mass spectrometry for a excessive answer snapshot and characterization of the virion, this unique e-book then keeps with capsid alterations and viral-like debris as promising choices to classical adenovirus vectors, and the learn of adenovirus in host interactions in vitro on the mobile point in addition to in vivo in animal types. eventually, the quantity concludes with an intensive replace of the best protocols to generate, magnify, and/or purify, at small and big scale, ordinary human Ad5 in addition to non-human, chimeric, and helper-dependent adenovirus vectors. Written within the vastly winning Methods in Molecular Biology sequence layout, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, effectively reproducible laboratory protocols, and tips about troubleshooting and averting recognized pitfalls.

Comprehensive and state of the art, Adenovirus tools and Protocols, 3rd Edition serves as a fantastic consultant for scientists carrying on with to analyze this hugely priceless viral tool.

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It is also possible to identify new proteins, encoded by the virus genome, attached or integrated in the virion. Today, liquid chromatography–mass spectrometry (LC-MS) is the method of choice for large-scale characterization of proteins, peptides, and PTMs. The so-called shotgun proteomics method provides unbiased protein identification and site determination of PTMs [10–12]. ), Adenovirus: Methods and Protocols, Methods in Molecular Biology, vol. 1007/978-1-62703-679-5_2, © Springer Science+Business Media, LLC 2014 25 26 Sara Bergström Lind et al.

4. Peptide extraction: Sonicate the gel pieces in the surrounding solution and collect the liquid. If the volume has dramatically decreased, add few more μL of 50 mM NH4HCO3 before sonication. Dry the liquid down in a SpeedVac and store the samples at −20 °C until analysis. 5 Enrichment of Phosphopeptides The TiO2 material is provided by the manufacturer packed in tips, Spin Tip (or can be produced in house [31]) and should be handled using a centrifuge. See Note 7 for specific enrichment of tyrosine phosphorylated peptides.

3. The virus now forms a band between the two CsCl densities (by the marker on the tube) that can be seen by eye when holding a black paper behind the tube. Carefully puncture the bottom of the tubes with a sharp-edged tool and let the liquid slowly drip out. Use a waste tube to collect most of the solution that drips before the virus close band. Then change to a clean Eppendorf tube and collect the virus band. 4. 34 g/cm3. Mix gently by pipetting up and down three to four times. 2. Ultracentrifugate (equilibrium gradient) at ~80,000 × g for 18–22 h at 4 °C.

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