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

NIH-3T3 cells were grown in 15 cm petri dishes under normal conditions up to 80% confluency and fixed with formaldehyde. Fixation, enzymatic shearing and immunoprecipitation were performed with the ChIP-IT Express Enzymatic Kit (Active Motif) according to the manufacturer’s instructions. Chromatin was precipitated with either a monoclonal ChIP grade antibody against TWIST1 (Abcam, ab50887) or anti CLOCK antibody (ab 3517). As positive control a monoclonal RNA Pol II antibody, as negative control a IgG antibody was used 10μg of sheared chromatin and 2μg of antibody was used in 100μl reaction volume. Eluted and reverse cross-linked chromatin was analyzed via qPCR of region of interest. Primers for E-box regions: Per1 E-box:
fw: 5’-GGGTAGTTTCCCTCCCTCAC-3’
rv: 5’- TGGCATCTGATTGGCTACTG-3’.
Dbp E-box:
fw: 5’-TCAACATGGTACAGCCCAGA-3’
rv: 5’-TGTGGGAGCTGAGCACATAG-3’.
The analysis method entailed solving for the DNA quantity of the ChIP and IgG samples, then calculating the fold enrichment of the ChIP sample relative to the IgG sample.

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Manufacturer protocol

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

Download manufacturer protocol

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