By Lucio Comai, Jonathan E. Katz, Parag Mallick
This quantity goals to supply protocols on a variety of biochemical equipment, analytical methods, and bioinformatics instruments constructed to investigate the proteome. Written within the hugely successful equipment in Molecular Biology series structure, chapters comprise introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, without problems reproducible laboratory protocols, and tips about troubleshooting and keeping off identified pitfalls.
Authoritative and state-of-the-art, Proteomics: tools and Protocols goals to make sure winning leads to the extra research of this very important field.
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Additional info for Proteomics: Methods and Protocols
6. Pool the pellets in one 50 mL conical tube (see Note 6). Centrifuge each time at 3200 × g for10 min at 4 °C. Cross-link the protein–DNA complexes by incubating the cells with formaldehyde diluted to a 3 % final concentration for 30 min at room temperature on a nutator (volume of the cross-linking solution must not be below 50 times the volume of pelleted cells: for a pellet of 4 mL cells, use at least 200 mL of cross-linking solution). Kan et al. 2. Centrifuge the cross-linked cells at 3200 × g for 10 min at 4 °C.
Discard the supernatant. Proteome Characterization of a Chromatin Locus… 27 8. Resuspend in 1 mL of low salt buffer. Incubate for 5 min at 42 °C in a thermomixer shaking at 500 rpm. 10. Immobilize the beads on the magnetic stand and discard the supernatant. 7 Elution 1. Resuspend the beads in 900 μL of elution buffer. Incubate for 30 min at 42 °C and an additional 10 min at 65 °C in the Thermomixer shaking at 500 rpm. 3. Immobilize the beads on the magnetic stand. 4. 5 mL microcentrifuge tubes.
Spare 10 μL for the input. 5 Hybridization and Chromatin Capture 1. Split the chromatin in two equal volumes, one for the experiment and the other for the scrambled control (see Note 17). 25 μM (see Note 18). Aliquot 150 μL of the chromatin-LNA mixture into PCR tubes. Kan et al. 4. Hybridize in a standard thermocycler using the following program (see Note 19): (a) 25 °C for 3 min (b) 71 °C for 9 min. (c) 38 °C for 1 h. (d) 60 °C for 3 min. (e) 38 °C for 30 min. (f) 60 °C for 3 min. (g) 38 °C for 30 min.