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

ChIP assays were carried out on flash-frozen mouse kidney or liver tissue using the ChIP-IT High-Sensitivity Kit (cat.53040 – Active Motif, Carlsbad, USA). The assay was carried out as per the manufacturer’s recommendations with slight modifications as described. Following formaldehyde-assisted chromatin fixation, tissue was homogenised using the handheld TissueRuptor (Qiagen). For kidney tissue, cells were then passed through a cell strainer in between PBS washes to obtain more uniform cell suspensions, due to the fibrosity of kidney tissue. Nuclei release was achieved by dounce homogenisation using a tight pestle. Chromatin was sheared by sonication using the EpiShear Probe Sonicator (Active Motif) with a 3.5 mm probe. Sonicating conditions were as follows: amplitude 37%, pulse for 30 s on and 30 s off, for a total sonication time of 2 min; this cycle was repeated eight times per sample. To prevent overheating and denaturation, samples were kept on ice between cycles, and the EpiShear-cooled sonication platform was used and replaced regularly throughout the sonication process.

Following sonication, input DNA was generated by treating samples with RNase A and proteinase K, heating, and then precipitating with ethanol. Input DNA concentrations were determined by both spectrophotometric (Nanodrop 2000 – Thermo Fisher Scientific) and fluorometric (Qubit HS or BR dsDNA assay – Thermo Fisher Scientific) quantification. A diluted aliquot of each input was electrophoresed by TapeStation (Agilent Technologies) to assess chromatin shearing efficacy.

Immunoprecipitation reactions were performed on prepared chromatin by incubation with antibodies for histone H3K27ac (cat.39133 – Active Motif), histone H3K27me3 (cat. 39155 – Active Motif) and GR (cat.240501AP – Proteintech Group, Rosemont, USA; cat.3660 (D8H2) – Cell Signaling Technology), on an end-to-end rotator overnight at 4°C. Incubation with MagReSyn protein G beads (cat.MR-PRG002 – ReSyn Biosciences, Gauteng, South Africa) on an end-to-end rotator was then performed for 3 h at 4°C. Beads (with antibody-chromatin complexes bound) were then washed, and ChIP material was eluted from the beads using Active Motif proprietary kit buffer (unless stated otherwise). ChIP DNA was treated with proteinase K and heated for 2.5 h to reverse cross-links, and then purified on a column using 36 μL proprietary Active Motif DNA purification elution buffer. For spike-in normalisation, spike-in (Drosophila melanogaster) chromatin (cat.53083 – Active Motif) and its specific antibody (cat.61686 – Active Motif) were added simultaneously to each IP reaction.

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Manufacturer protocol

Download the product protocol from Active Motif for ChIP-IT High Sensitivity® below.

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