ChIPAb+ Trimethyl-Histone H3 (Lys4) - ChIP Validated Antibody and Primer Set, rabbit monoclonal

All ChIPAb+ antibodies are individually validated for chromatin precipitation, every lot, every time. Each ChIPAb+ antibody set includes control primers (tested every lot by qPCR) to biologically validate your IP results in a locus-specific context. The qPCR protocol and primer sequences are provided, allowing researchers to validate ChIP protocols when using our antibody in their chromatin context. Each set also includes a negative control antibody to ensure specificity of the ChIP reaction.
The ChIPAb+ Trimethyl-Histone H3 (Lys4) set includes the anti-trimethyl-histone H3 (Lys4) antibody, a negative control antibody (normal rabbit serum), and qPCR primers which amplify a 166 base pair region within the promoter of the human GAPDH gene. The trimethyl-histone H3 (Lys4) and negative control antibodies are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of trimethyl-histone H3 (Lys4)-associated chromatin.

The methylation of histones can occur on two different residues: arginine or lysine. Histone methylation can be associated with transcriptional activation or repression, depending on the methylated residue. Lysine 4 of histone H3 can be mono-, di- or trimethylated by different histone methyltransferases (HMTs) such as SET1 or ASH1. Methylation of Lys4 is often associated with transcriptional activation. The demethylase LSD1 is able to demethylate histone H3 Lys4.

Trimethyl-Histone H3 (Lys4)

Epitope: Trimethyl Lys4

The trimethyl-histone H3 (Lys4) antibody is made against a BSA-conjugated, synthetic peptide containing the sequence …RT[me3K]QT… in which me3K corresponds to trimethyl-lysine 4 of human histone H3

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 Category
Epigenetics & Nuclear Function

Research Sub Category
Chromatin Biology

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.

25 assays per kit, ~3μL per chromatin immunoprecipitation

Anti-Trimethyl-Histone H3 (Lys4) (purified rabbit IgG), 1 vial

Negative ChIP Control Rabbit IgG, 1 vial

ChIP Primers GAPDH, 1 vial

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from untreated HeLa cells (1 X 10⁶ cell equivalents) was subjected to chromatin immunoprecipitation using 3 μL of either a normal rabbit IgG or 3 μL Anti-Trimethyl-Histone H3 (Lys4) Monoclonal IgG and the Magna ChIP A (Part # 17-610) Kit. Successful immunoprecipitation of
trimethyl-histone H3 (Lys4) associated DNA fragments was verified by qPCR using control ChIP Primers flanking the human GAPDH promoter (Please see figures). Please refer to the EZ-Magna A ChIP protocol for experimental details.

17 kDa

Anti-Trimethyl-Histone H3 (Lys4) recombin-ant rabbit monoclonal IgG. One vial containing 75 μL of protein A purified rabbit IgG in storage buffer (0.1M Tris-Glycine pH 7.4, 0.15M NaCl, 0.05% NaN3, with the addition of 40% glycerol.

Normal Rabbit IgG. One vial containing 75 μL of normal rabbit IgG.

Control Primers. One vial containing 75 μL of 5 μM of each primer specific for human GAPDH.
FOR: TAC TAG CGG TTT TAC GGG CG
REV: TCG AAC AGG AGG AGC AGA GAG CGA

Format: Purified

Stable for 1 year at -20°C from date of receipt

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

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