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

ChIP was performed using the ChIP-IT Express and control kits (Active Motif, Carlsbad, California) according to the manufacturer's instructions. In brief, after cross-linking, cells were homogenized in cell lysis buffer and then sonicated on ice using a Fisher Scientific Sonic Dismembrator 100 (output control 4, duty cycle 40%; Fisher Scientific, Pittsburgh, Pennsylvania) to an average length of approximately 500 bp as determined by 2% agarose gel electrophoresis. A total of 100 μL of sheared cross-linked chromatin for each ChIP reaction was immunoprecipitated overnight at 4°C with 25 μL of protein G magnetic beads, 2 μg of antimethyl-CpG binding protein 2 (Mecp2) antibody (Millipore), and negative IgG and anti-RNA Pol II IgG (positive control) (Active Motif). After IP, chromatin was eluted, and the DNA and histones/other proteins were dissociated by reverse cross-linking. Finally, the dissociated proteins were digested with proteinase K, and the eluted DNA was subjected to PCR.

SeqChIP was performed according to Geisberg and Struhl (16) with modifications of Furlan-Magaril et al (17). After the DNA-chromatin complex was immunoprecipitated by the anti-Mecp2 antibody and dissociated from the secondary antibody linked protein G magnetic beads, re-IP was carried out with the anti-Creb1 antibody (Santa Cruz Biotechnology, Inc, Santa Cruz, California) or anti-HDAC1 antibody (ChIP validated; Millipore) supplemented with magnetic G beads, following the identical procedure described above.

To quantify protein/histone-associated DNA, ChIP-quantitative PCR (qPCR) reactions were performed using SYBR GreenER Assay (Invitrogen) with the eluted DNA serving as the template (18). Protein enrichment was expressed as the ratio of IP-DNA/input (target) to IP-DNA/input (RNA PoI II) followed by standardization to an internal control (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]).

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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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