ChIPAb+ Trimethyl-Histone H3 (Lys4)

Stock Code: 3586450
Manufacturer Part No: 17-614

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Descriptions

Analysis Note

ControlIncluded negative control antibody purified rabbit IgG and control primers specific for human GAPDH promoter.

Application

Trimethyl-Histone H3 (Lys4) ChIP validated antibody & primer set including the ChIP-grade antibody & the specific control PCR primers used for chromatin immunoprecipitation of H3K4Me3.

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from untreated or UV treated (6 hrs, 50 joules/m2.) U2OS cells (3 X 10⁶ cell equivalents per IP) was subjected to chromatin immunoprecipitation using 3 μL of rabbit Anti-Trimethyl Histone H3 (Lys4) and the Magna ChIP™ A (Cat. # 17-610) Kit. Immunoprecipitation of trimethyl histone H3 (Lys4) associated DNA fragments was verified by qPCR using ChIP Primers p21 flanking the human p21 promoter that contains an Sp1 binding site (Please see figures).
Fold Increase is a ratio of normalized mean IP quantities extracted from standard curves derived from inputs of each chromatin sample. Trimethyl-histone (Lys4) immunoprecipitable activity associated with this promoter increases with UV treatment as observed in other studies.
Please refer to the EZ-Magna A ChIP™ (Cat. # 17-408) or EZ-ChIP™ (Cat. #17-371) protocol for experimental details.

ChIP-seq Analysis:
Chromatin immunoprecipitation was performed using the Magna ChIP™ HiSens kit (17-10460), 3 µL anti-trimethyl-Histone H3 (Lys4) antibody (cat# 17-614) or, 20 µL Protein A/G beads, and 1e6 crosslinked HeLa cell chromatin followed by DNA purification using magnetic beads. Libraries were prepared from Input and ChIP DNA samples using standard protocols with Illumina barcoded adapters, and analyzed on Illumina HiSeq instrument. An excess of eighteen million reads from FastQ files were mapped using Bowtie (http://bowtie-bio.sourceforge.net/manual.shtml) following TagDust (http://genome.gsc.riken.jp/osc/english/dataresource/) tag removal. Peaks were identified using MACS (http://luelab.dfci.harvard.edu/MACS/), with peaks and reads visualized as a custom track in UCSC Genome Browser (http://genome.ucsc.edu) from BigWig and BED files. The highest 25% of peaks identified in the 17-614 and 07-473 datasets showed 99% overlap with peaks identified in the ENCODE H3K4me3 BROAD Histone track for HeLa S3.

Western blot analysis and peptide inhibition:
Representative blot. HeLa acid extract was resolved by electrophoresis, transferred to nitrocellulose and probed with anti-trimethyl-histone H3 (Lys4) (1:2,000, lane 1) or preincubated with 0.4 μM Histone H3 peptide with following modifications Lane 2: monomethyl-Lysine 4, Lane 3: dimethyl Lysine 4, Lane 4: trimethyl-Lysine 4.
Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection
system (Please see figures).

Research CategoryEpigenetics & Nuclear Function

Research Sub CategoryChromatin Biology

Components

Anti-Trimethyl-Histone H3 (Lys4) (purified rabbit IgG), 1 vialNegative ChIP Control Rabbit IgG, 1 vialChIP Primers GAPDH, 1 vial

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