ChIP-IT High Sensitivity®

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Do not re-freeze the Protein G magnetic beads, the beads should be stored at 4C.	-Always use ChIP validated antibody. -Optimal use of 1-3 ug of antibody.	-The diluted Proteinase K stop solution can not be stored.

Upstream tips
-Do not re-freeze the Protein G magnetic beads, the beads should be stored at 4C.
Protocol tips
-Always use ChIP validated antibody. -Optimal use of 1-3 ug of antibody.
Downstream tips
-The diluted Proteinase K stop solution can not be stored.

Publication protocol

A large part of the cerebral cortex (from Bregma +1 mm to posterior end) was dissected from P28 C57BL/6J mice and flash-frozen in liquid nitrogen. Pooled cortices from 10 males and 10 females (total 20 mice) were cross-linked with 1% formaldehyde in PBS for 15 min at room temperature and ChIP was performed using the ChIP-IT High Sensitivity kit (Active Motif) per the manufacturer's instructions, with some modifications: after homogenization using a Dounce homogenizer, nuclei were washed in LB0 buffer (20 mM Tris-HCl [pH7.5], 10 mM NaCl, 1 mM EDTA, 0.2% NP-40, 1 mM PMSF) and resuspended in MNase buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 2.5 mM MgCl2, 0.1% NP-40, 1 mM DTT, 1 mM PMSF), provided with cOmplete™ EDTA-free protease inhibitor cocktail (Roche Diagnostics). Sonication was carried out using a Misonix 2000 Sonicator (Misonix) at power 7. After four pulses of 25 s each, 5 mM CaCl2 and MNase (NEB, 3,000 U in 500 μl reaction mixture) were added and incubated for 10 min at 37°C. The reaction was stopped by adding 20 mM EGTA. The samples were diluted by half with 2 × ChIP dilution buffer (2% Triton X-100, 280 mM NaCl, 80 mM Tris-HCl [pH8.0] in ChIP buffer from the Active Motif kit) and further sonicated for two pulses of 25 s each. Insoluble material was removed by centrifugation at 15,000 rpm for 5 min at 4°C. Fragmented chromatin sample was divided into four aliquots, of which three were immunoprecipitated with three independent anti-Otx2 antibodies: CH (Millipore AB9566, 4 μl polyclonal rabbit serum), R&D (R&D systems AF1979, 4 μg polyclonal goat IgG) and Operon2 (generated in this study, 6 μg polyclonal rabbit IgG raised against full-length Otx2). Normal rabbit IgG (Abcam ab46540) and normal goat IgG (Santa Cruz sc-2028) were used as negative controls. For immunoprecipitation with CH antibody only, which is not affinity-purified, chromatin was pre-cleared by incubating with protein G agarose-bound normal rabbit IgG for 1 h to increase the signal-to-noise ratio. Samples were assessed for enrichment by qPCR with SsoAdvanced Universal SYBR Green SuperMix (BioRad).

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Manufacturer protocol

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