Chromatin and Chromatin Remodeling Enzymes by Wu L.A., Allis C.D.

By Wu L.A., Allis C.D.

DNA within the nucleus of plant and animal cells is kept within the type of chromatin. Chromatin and the Chromatin remodelling enzymes play a tremendous position in gene transcription.

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Use the circular dialysis membrane (see Materials). 2 Â TBE (2000 ml) at 4 and at a power of 10 W. Record a baseline at 260 nm, using TCS buffer (TCS buffer used during reconstitution can be reused for this purpose) as elution buffer. 2. Concentrate NCP (in TCS buffer) to a maximum of 600 l for a 4-mg reconstitution. Mix with sucrose to a final concentration of 5% (v/v) 44 biochemistry of histones, nucleosomes, and chromatin [3] and load on the preparative gel, using a syringe with an attached piece of tubing.

Extinction coefficients can be [2] reconstitution of NCP 37 approximated by adding up those of individual histones (times two). Yields of pure histone octamer are usually between 50 and 75% of the input material (yields may be lower for octamer containing tailless histones).  7. Octamer can be stored on ice (for short-term storage), or at À20 as a 50% (v/v) glycerol solution (for long-term storage). Octamer containing globular histones usually is stable only for a few months and can often form higher order aggregates on storage for longer periods of time.

Remove the contents from the dialysis buttons, incubate one-third  each for 2 h at 37 and 55 , respectively (spin frequently to collect condensation), and leave one-third on ice. 4. Analyze the products by using the high-resolution gel-shift assay described earlier1 and later in the chapter. Sample that has not been heat treated is run as a control. For subsequent large-scale reconstitutions, choose conditions that (1) contain only a small (if any) excess of free DNA and (1) are completely shifted to a single position.

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