Immunoprecipitation

1. Recover supernatants after centrifugation and transfer to a new tube. Dilute samples 10-fold using ChIP dilution buffer (see Note 5). Samples will then be in conditions suitable for immunoprecipitation.

Fig. 2. Example of sonicated islet DNA of a suitable size for immunoprecipitation. Approximately 100 islets where sonicated in 150 iL of SDS lysis buffer for six 30-s pulses with the sonifier microtip at maximum output. Crosslinks were reverted and DNA was recovered as described in the text and analyzed by electrophoresis in a 1% agarose gel stained with ethidium bromide. Fragment size ranges from 0.4 to 2 kb.

Fig. 2. Example of sonicated islet DNA of a suitable size for immunoprecipitation. Approximately 100 islets where sonicated in 150 iL of SDS lysis buffer for six 30-s pulses with the sonifier microtip at maximum output. Crosslinks were reverted and DNA was recovered as described in the text and analyzed by electrophoresis in a 1% agarose gel stained with ethidium bromide. Fragment size ranges from 0.4 to 2 kb.

2. Preclear samples prior to immunoprecipitation by incubating every 0.5 mL with 30 iL salmon sperm DNA/protein A for 1 h at 4°C with gentle rotation.

3. Centrifuge samples and recover supernatant in new Eppendorf tubes. Generally use 0.5 mL for each individual immunoprecipitation.

4. Add antibody: 1-2 ig for antiacetylated histone H3 or H4 (UBI) and for other monoclonal or polyclonal affinity purified antibodies, an empirical amount for other serums. Add preimmune IgGs or unrelated serum to one sample, which will be the negative control to check for specificity of the assay (see Note 6).

5. Incubate overnight at 4°C on a rotating wheel.

6. Centrifuge samples at maximum speed in microfuge for 2 min and transfer super-natants to new tubes. This step is necessary to precipitate and discard any aggre gates that may have formed during the overnight incubation and that would otherwise be nonspecifically precipitated in the following steps.

7. Add 30 iL salmon sperm DNA/protein A to each 500 iL sample and continue incubation for 1-3 h at 4°C, to allow for the antibody-protein A complexes to form.

8. Centrifuge samples 2 min in a microfuge at maximum speed and discard super-natants. The supernatant of the control immunoprecipitation (performed with nonimmune or unrelated serum) can be kept to be used as input DNA control (see Note 7) for the PCR analysis of the experiment.

9. Wash pellets with 1 mL low-salt immune complex wash buffer for 3-5 min at room temperature on rotating wheel. Centrifuge 2 min in a microfuge at maximum speed and discard supernatants.

10. Wash pellets with 1 mL high-salt immune complex wash buffer for 3-5 min at room temperature on rotating wheel. Centrifuge 2 min in microfuge at maximum speed and discard supernatants. These washing steps are intended to eliminate nonspecific protein-antibody interactions.

11. Wash pellets with 1 mL LiCl immune complex wash buffer for 3-5 min at room temperature on a rotating wheel. Centrifuge 2 min in microfuge at maximum speed and discard supernatants. This washing step eliminates low-affinity protein-antibody interactions.

12. Wash pellets three times with 1 mL TE buffer for 3-5 min at room temperature on a rotating wheel to eliminate excess LiCl in the samples. Centrifuge 2 min in microfuge at maximum speed and discard supernatants.

13. Elute samples by adding 200 iL elution buffer prepared fresh and incubating for 15 min at room temperature on a rotating wheel.

14. Centrifuge 2 min in microfuge at maximum speed. Recover supernatants and transfer to new tubes. Add 200 iL elution buffer to the beads and repeat elution for another 15 min.

15. Centrifuge 2 min in a microfuge at maximum speed. Recover supernatant and pool with the first elution (total 400 iL per sample). The eluate contains the selected proteins with the associated DNA.

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