EZ-Magna ChIP™ G - Chromatin Immunoprecipitation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- DO NOT cryo-preserve specimen with sucrose. -Make fresh 1% formaldehyde before each experiment.	-Keep lysate ice-cold. Sonication produces heat, which can denature the chromatin. Allow time between cycles of sonication to prevent sample overheating. - DNA fragments should be in 200 -1000 bps in size. -Add 1-10 µg of immunoprecipitating antibody.

Upstream tips
- DO NOT cryo-preserve specimen with sucrose. -Make fresh 1% formaldehyde before each experiment.
Protocol tips
-Keep lysate ice-cold. Sonication produces heat, which can denature the chromatin. Allow time between cycles of sonication to prevent sample overheating. - DNA fragments should be in 200 -1000 bps in size. -Add 1-10 µg of immunoprecipitating antibody.

Publication protocol

Whole ovaries were fixed with 1% formaldehyde (Sigma) in Leibovitz’s L-15 medium (Life Technologies) for 30 minutes. An EZ- Magna ChIPTM G kit (Millipore) was used according to manufacturer’s instructions for the preparation of nuclear extracts and subsequent ChIP. 2% of the initial volume was taken as control for the IPs (Input). Protein G magnetic beads (Merck-Millipore) were added to each aliquot along with 1 µg of immunoprecipitating antibody against SMAD2/3 (sc-133098, Santa Cruz) and non-immune mouse IgG (I-200, Vector Laboratories). As a positive control, an antibody against the protein FOXL2 was used (0.2 µg/ml; sc-55655, Santa Cruz), since Ccnd2 and p27 are known targets of this protein56,57. An equal volume (4 µl) of IP sample, control or RNAse/DNAse free water (additional negative control) were analysed by qPCR in duplicate using primers designed to flank predicted FOXL2, SMAD3 and MYC binding sites (TRANSFAC; Table S1) in 384 well-plates as described above. Input, IPs and IgG control samples were analysed using the ‘% input’ method where the mean Ct value of each IP and IgG sample was normalised against the input sample. Since the starting input sample was 2% of the initial chromatin, a dilution factor of 50 or 5.64 cycles (log 2 of 50) was subtracted and calculations were performed as follows: i) Mean Ct INPUT – 5.64, ii) Mean Ct IP (or IgG) - Mean Ct INPUT = ΔCt, iii)% INPUT = (2 (−ΔCt)) × 100.

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Manufacturer protocol

Download the product protocol from Merck Millipore for EZ-Magna ChIP™ G - Chromatin Immunoprecipitation Kit below.

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