Chromatin IP (CHIP assay)

This protocol has some minor modification to the protocol described in Strahl-Bolsinger S. et al. [1997, Gen & Dev 11, p83-93] and was obtained from Flick K. (The Scripps Research Institute).

• use 50 ml cells OD_600nm = 0.7 - 1.0 per timepoint / sample
• add 1.35 ml 37 % Formaldehyde (endconcentration = 1 %), incubate 15 min at 25 °C
• add 2.5 ml 2.5 M Glycine, incubate 5 min at 25 °C
• spin down, wash once with 20 ml Pbs
• transfer to 2 ml Eppendorf tube, wash again with Pbs and freeze cells or proceed on
• resuspend in 200 - 400 µl CHIP lysis buffer, add an equal volume of glass beads
• shake for 30 min at 4 °C on vortexer (maximum level)
• pierce tube bottom with needle and spin liquid into fresh tube
• resuspend extracts and sonicate for 30 sec level 2 (Branson, microtip probe)
• spin extract for 10 min, 10,000 rpm, at 4 °C
• take supernatant and measure protein concentration (BioRad assay)
• use 1 - 5 mg protein per IP
• immunoprecipitate for 2 h to ON, 4 °C
• wash immunoprecipitations pelleting the beads each time:
       2 x 1 ml CHIP lysis buffer
       2 x 1 ml CHIP lysis buffer (high salt))
       2 x 1 ml CHIP wash buffer
       2 x 1 ml TE
• elute immunoprecipitations: add 75 µl elution buffer
• incubate for 10 min at 65 °C
• spin, take supernatant, elute pellet again with 75 µl elution buffer
• combine supernatants, incubate at 65 °6 h to ON
• take 1/100 of the protein amount taken for the IP, add to 150 µl elution buffer (INPUT control)
• incubate at 65 °C 6 h to ON
• add 750 µl PB buffer (Qiagen PCR purification kit)
• purify DNA through Qiaquick column
• elute DNA into 50 µl H₂O
• use 0.5 - 1 µl per 25 µl PCR reaction:
  
       1 x 95 °C, 2 min
       21 x 95 °C, 30 sec; 60 °C, 30 sec; 72 °C, 1 min
       1 x 72 °C, 3 min
  
• primers (1 µM): 20 - 24 bp, 50 % GC, producing a 200 - 500 bp fragment
• PCR products (10 - 15 µl) are separated on 2 % agarose gels or 5 -6 % non denaturing PAA gels
  

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Buffers:

 2.5 M Glycine
[for 500 ml: 93.84 g]

 Pbs:
[for 1l: 8 g NaCl; 0.2 g KCl; 1.15 g Na₂HPO₄ * 7H₂O; 0.2 g KH₂PO₄]

 CHIP lysis buffer:
50 mM HEPES pH 7.5; 140 mM NaCl; 1 % Triton X100; 0.1 % NaDeoxycholate; protease inhibitors.
[ for 500 ml: 25 ml 1 M HEPES pH 7.5; 18 ml 4 M NaCl; 50 ml 10 % Triton X100; 5 ml 10 % NaDeoxycholate; protease inhibitors]

 CHIP lysis buffer (high salt):
50 mM HEPES pH 7.5; 500 mM NaCl; 1 % Triton X100; 0.1 % NaDeoxycholate; protease inhibitors.
[ for 500 ml: 25 ml 1 M HEPES pH 7.5; 62.5 ml 4 M NaCl; 50 ml 10 % Triton X100; 5 ml 10 % NaDeoxycholate; protease inhibitors]

 CHIP wash buffer:
10 mM Tris pH 8.0; 250 mM LiCl; 0.5 % NP-40; 0.5 % NaDeoxycholate; 1 mM EDTA.
[ for 500 ml: 5 ml 1 M Tris pH 8.0; 5.3 g LiCl; 25 ml 10 % NP-40; 25 ml 10 % NaDeoxycholate; 1 ml 0.5 M EDTA]

 elution buffer:
50 mM Tris pH 8.0; 1 % SDS; 10 mM EDTA.
[ for 10 ml: 0.5 ml 1 M Tris pH 8.0; 1 ml 10 % SDS; 0.2 ml 0.5 M EDTA]