By Lucio Comai, Jonathan E. Katz, Parag Mallick
This quantity goals to supply protocols on a variety of biochemical tools, analytical methods, and bioinformatics instruments constructed to investigate the proteome. Written within the hugely successful tools in Molecular Biology series structure, chapters comprise introductions to their respective issues, lists of the required fabrics and reagents, step by step, simply reproducible laboratory protocols, and tips about troubleshooting and averting identified pitfalls.
Authoritative and state of the art, Proteomics: tools and Protocols goals to make sure profitable leads to the additional research of this very important field.
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A) Venn diagram of proteins identified by LC-MS/MS in the nuclear and cytoplasmic enriched fractions of MCF10A cells. The overlap between nuclear enriched and cytoplasmic enriched fraction (55 %) as well as the number of proteins identified in only one of those fractions (45 %) shows the complementarity of both subcellular proteomic profiles. (b) Gene Ontology classification of proteins enriched in the nuclear and cytoplasmic compartments also supports the complementarity of the proteomic profiles 6.
Ide S, Dejardin J (2015) End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter. Nat Commun 6:6674 4. Belotserkovskii BP, Reddy G, Zarling DA (1999) DNA hybrids stabilized by heterologies. Landgraf R, Chen CH, Sigman DS (1995) R-loop stability as a function of RNA structure and size. Nucleic Acids Res 23:3516–3523 7. Hirsch JD, Eslamizar L, Filanoski BJ, Malekzadeh N, Haugland RP, Beechem JM, Haugland RP (2002) Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation.
3. Incubate on ice for 10 min. 4. Centrifuge at 16,000 × g for 10 min at 4 °C. Carefully remove the supernatant with a pipette and leave about 50 μL (see Note 26). 6. Wash the pellet twice with 1 mL of ice-cold acetone 100 %. 7. Vortex for 10 s. 8. Centrifuge at 16,000 × g for 10 min at 4 °C. 9. Carefully remove the supernatant and leave about 50 μL. 10. Evaporate the acetone by heating the tube at 100 °C for 10 s. Repeat this step if the acetone is not entirely evaporated. Resuspend in 70 μL of cross-linking reversal solution if the sample will be analyzed by mass spectrometry or in 100 μL of reversal cross-linking solution if the sample is used for western- blot analysis.