ChIP-IT® Express Chromatin Immunoprecipitation Kits

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

Chromatin Immunoprecipitation assay was performed using the Chip-IT Express Kit (Active Motif) as previously described (13, 26). Briefly, tissues were minced and protein-DNA complexes were fixed with 1.5% formaldehyde. Tissues were then homogenized in lysis buffer (10 μg/mL Leupeptin, 10 μg/mL Aprotinin, and 1 mM PMSF) on ice. Samples were sonicated to shear chromatin to an average length between 200 and 1,000 bp. After incubation with antibodies against CREB (binding site at – 4408 and – 3896) (#4820; Cell Signaling, Danvers, MA, United States) or Sp1 (binding site at – 3425 and – 3034) (39058; Active Motif), magnetic beads (50 μl) were added into the samples. Crosslinking was then reversed using a salt solution and proteins were digested with proteinase K. The antibody-pulled chromatin extracts were then subjected to real-time quantitative PCR analysis using two primers that flank the predicted transcription factor binding sites at GR promoters, as described above in MeDIP.

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Manufacturer protocol

Download the product protocol from Active Motif for ChIP-IT® Express Chromatin Immunoprecipitation Kits below.

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