Isolation and fractionation of nuclei



All steps on ice or at 4°C. To all HEPES buffers used 1x protease inhibitor, 1 mM DTT and 0.1 mM PMSF should be added freshly before use. For the sample, please calculate the number of embryo equivalents (1 mg corresponds to about 100 embryos).

Isolation of nuclei

  • 10-100 mg of dechorionated embryos are resuspended in 1 ml HEPES buffer A
  • Douncing 5 to 10 times, 30 µl should be saved as the fraction Total + 10 µl 4x Laemmli
  • Centrifuge at 4000 rpm for 10 min (Eppendorf, table top centrifuge)
  • The supernatant should centrifuged at 14000 rpm, 10 min to remove lipids and saved as the fraction cytoplasm (30 µl + 10 µl 4x Laemmli)
  • Resuspend the pellet in 1 ml HEPES buffer A
  • Layer the solution carefully on top of 5 ml HEPES buffer B in a 15 ml Falcon tube
  • Centrifuge at 4000 rpm, 5 min (use a swing-out rotor!!)
  • Discard the supernatant, nuclei are in the pellet.
  • Resuspend the pellet in 1 ml HEPES buffer A
  • Layer the solution carefully on top of 5 ml Hepes Buffer B in a 15 ml Falcon tube
  • Centrifuge at 4000 rpm, 5 min (swing-out rotor!!)
  • Discard the supernatant
  • Once more resuspend the pellet in 1 ml Hepes Buffer A and centrifuge at 4000 rpm, 10 min). You may repeat the washing step upto three times.
  • Save a sample : 30 µl + 10 µl 4x Laemmli

Fractionation of nuclei

  • Resuspend the nuclei in 1 ml HEPES buffer C
  • Incubate for 15 min in ice
  • Centrifuge at 4000 rpm, 15 min
  • Save the supernatant as the fraction chromatin unbound fraction (CUF)
  • Resuspend the pellet in HEPES buffer A + 300 mM NaCl
  • Incubate for 5 minutes on ice
  • Centrifuge at 3000 rpm for 5 min
  • Save the supernatant as the fraction membrane bound proteins 300 (MBP 300)
  • Repeat salt washing steps with 600, 1200 mM or other concentrations of NaCl
  • Resuspend the last pellet in 1 ml Hepes Buffer A and save it as the fraction salt resistant pellet (SRP)

Protein precipitation using Trichloric acid (TCA)

  • All fractions from the fractionation steps should be precipitated using trichloroacetic acid
  • Add TCA to 15% (150 µl in 1 ml sample)
  • Incubate at -20°C for 5 min or over night
  • Incubate for 15 min on ice
  • Centrifuge at 14000 rpm, 10 min
  • Resuspend the pellet in 500 µl ice-cold acetone
  • Centrifuge at 14000 rpm, 10 min
  • Repeat acetone washing step
  • Resupend the pellet in an appropriate volume of Laemmli (e.g 30 µl 2x SB)
  • If necessary add a droplet of 1 M Tris pH 8 to adjust the pH.
  • Boil samples at 95°C 5 minutes

Buffers:

HEPES buffer A

    15 mM Hepes/KOH pH 7.4
    5 mM MgCl2
    350 mM Sucrose
    10 mM KCl

HEPES buffer B

    15 mM Hepes/KOH pH 7.4
    5 mM MgCl2
    800 mM Sucrose
    10 mM KCl

HEPES buffer C

    15 mM Hepes/KOH pH 7.4
    5 mM MgCl2
    350 mM Sucrose
    10 mM KCl
    1% Triton X-100

Protease cocktail (Roche)

Dissolve one tablet in 10 ml of lysis buffer. You do not need to add protease inhibitors to the washing buffer. The tablet may also be dissolved in 1.5 ml of water as a 7X stock solution. Please refer to the product description for full information.