Isolation of chromatin from egg extract and histone recovery (Shechter lab)

Protocol submitted by VGP from David Shechter lab protocols [1]

Link to PDF: [2]

PROTOCOL: Sperm Chromatin Isolation / Histone Preparation

Recipes:

ELB-CIB (Egg lysis buffer – Chromatin Isolation Buffer) - 1X Egg Lysis Buffer, containing .25M Sucrose 1mM Spermidine 1mM Spermine 0.5mM EDTA 5mM DTT 0.1% Triton-X 100 1X Protease Inhibitor Cocktail (Roche) 10mM Sodium Butyrate 1X Phosphatase Inhibitor Cocktails I & II (Roche)

ELB - 0.25M Sucrose 2.5mM MgCl2 50mM KCl 10mM Hepes-KOH pH 7.8 1mM DTT

Procedures/Steps:

1. Incubate sperm in egg extract (LSS, HSS, NPE, etc), use 10-100 μl/reaction, and 2000- 10,000 sperm/ μl. Flash freeze at time points or continue with unfrozen extract. For large-scale histone preparation: use 1-2ml of LSS extract and 10,000 sperm/ μl.

2. Add 900 μl of 1X ELB-CIB (ice-cold); mix thoroughly and incubate on ice for 10 minutes. Underlayer carefully with 200 μl of ELB-CIB containing 0.6M sucrose.

3. Spin in swinging bucket rotor at 4000g for 5-10 minutes at 4C. (In eppendorf, 6000RPM). There should be a visible white pellet at the bottom of the tube.

4. Carefully remove supernatant with pipet and respin briefly. Aspirate remaining supernatant with pipet.

5. Wash pellet 1X with 1ml ELB-CIB. (For histone prep, include 0.3M NaCl). Completely aspirate supernatant with pipet.

6. For gel analysis: Resuspend pellet in 1X Laemmli loading buffer. The DNA can get viscous when hot; cool before loading on SDS-PAGE gel.

7. For histone prep: Extract pellet in 0.4N H2SO4 for 2 hours or overnight. Precipitate extracted proteins in supernatant with 25% cold TCA for 30 minutes on ice, spin down and wash pellet with 100% cold acetone. Dissolve pellet in appropriate volume of water.