Chromatin Immuno-precipitation

Dictyostelium ChIP (Chromatin Immuno-precipitation) protocol

Contributed by Jeff Williams, Y. Yamada, and with grateful thanks to Jonathan Chubb for his advice when establishing the method, September 2005.


[MATERIALS] - [INDEX]

Procedure

Fixation

  1. Suspend about 5x108 cells in KK2 to 3.1ml. Add 0.4 ml of 10xPBS and 0.5 ml of 8% paraformaldehyde.
    Rotate for 2 hours at 4°C.

  2. Add glycine to 125 mM (0.25 ml of 2M), agitate at room temperature for 5 min.

  3. Wash with PBS 4 times and with TBS once. (Can freeze at -80°C here.)

Lysis and sonication

  1. Suspend cells in TBS. Add the same volume of 2xTTS/1xTBS and protease inhibitors.
    Mix and leave on ice for 15-20 min.

  2. Sonicate DNA down to 0.3-2.5 kb. (9 sec, 5 times with Branson sonifier, output 6)

  3. Centrifuge at 14k rpm for 10 min. Transfer sup into a fresh tube and spin again for 20 min. Take sup.

IP and DNA prep

  1. Preabsorption:
    • Dilute cell lysate twice with TTS buffer.
    • Add protein A Sepharose CL-4B (40 µl of 1:3 slurry) and incubate at 4°C for a few hours.

  2. Centrifuge at a top speed for 5 min. Take sup. Keep an aliquot (20 µl) as an input fraction.

  3. To cell lysate (1x107 cells, or 400 ng protein) add 40 µl of 10% Blocking Reagent, protease inhibitors and top up with TTS buffer to 0.8 ml.

  4. Add antibody and incubate at 4°C overnight.

  5. Add 80 µl of 1:3 slurry of Protein A Sepharose CL-4B and incubate at 4°C for several hours.

  6. Wash with TTS, 4 times (Spin at a low speed (6000-7000rpm), 1min.

  7. Wash with LiDN buffer, 2 times.

  8. Spin at a top speed for 1 min and remove the remaining sup.

  9. Add 100 µl of E-buffer. Incubate at 37°C for 15 min.

  10. Spin and take 90 µl of sup.

  11. Incubate at 65°C overnight to reverse crosslink. To input fraction, add E-buffer and do the same.

  12. Add 90 µl TE containing Proteinase K (200 mg/ml) and RNase (20 mg/ml). Incubate at 37°C for 1 hour.

  13. Purify DNA with QIAquick PCR purification column. Elute with 40-50 µl EB.

  14. Analyse by PCR.

[TOP] [INDEX]


Materials

  • 2xTTS/1xTBS
    • 50 mM Tris pH8.0
    • 150 mM NaCl
    • 2% Triton X100
    • 4 mM EDTA
    • 0.1% SDS


  • TTS
    • 50 mM Tris pH 8.0
    • 150 mM NaCl
    • 1% Triton X100
    • 2 mM EDTA
    • 0.05% SDS


  • LiDN buffer
    • 10 mM Tris pH8.0
    • 250 mM LiCl
    • 1% DOC
    • 1% NP-40
    • 1 mM EDTA


  • E-buffer
    • 50 mM Tris pH7.5
    • 1% SDS
    • 1 mM EDTA


[TOP] [INDEX]

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