H3K4me3 Polyclonal Antibody

$HeLa cells were stained with the Bioss antibody against H3K4me3 (Cat. No. bs-53103R) and with DAPI. Cells were fixed with 4% formaldehyde for 10\u2019 and blocked with PBS\/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H3K4me3 antibody (left) diluted 1:100 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.$

HeLa cells were stained with the Bioss antibody against H3K4me3 (Cat. No. bs-53103R) and with DAPI. Cells were fixed with 4% formaldehyde for 10\u2019 and blocked with PBS\/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H3K4me3 antibody (left) diluted 1:100 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

$Western blot was performed on whole cell (40 \u00b5g, lane 1) and histone extracts (15 \u00b5g, lane 2) from HeLa cells, and on 1 \u00b5g of recombinant histone H2A, H2B, H3.1, H3.3 and H4 (lane 3, 4, 5, 6 and 7, respectively) using the Bioss antibody against H3K4me3 (Cat. No. bs-53103R). The antibody was diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is shown on the right, the marker (in kDa) is shown on the left.$

Western blot was performed on whole cell (40 \u00b5g, lane 1) and histone extracts (15 \u00b5g, lane 2) from HeLa cells, and on 1 \u00b5g of recombinant histone H2A, H2B, H3.1, H3.3 and H4 (lane 3, 4, 5, 6 and 7, respectively) using the Bioss antibody against H3K4me3 (Cat. No. bs-53103R). The antibody was diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is shown on the right, the marker (in kDa) is shown on the left.

A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K4me3 (Cat. No. bs-53103R) with peptides containing other modifications and unmodified sequences of histone H3 and H4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:2,000. The figure shows a high specificity of the antibody for the modification of interest.

$ChIP was performed on sheared chromatin from 1 million HeLa cells using 1 \u00b5g of the Bioss antibody against H3K4me3 (Cat. No. bs-53103R) as described above. The IP'd DNA was subsequently analyzed on an Illumina Genome Analyzer. Library preparation, cluster generation, and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. The figure shows the peak distribution along the complete sequence and a 1.2 Mb region of the X-chromosome (figure A and B) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure C and D). These results clearly show an enrichment of the H3K4 trimethylation at the promoters of active genes.$

ChIP was performed on sheared chromatin from 1 million HeLa cells using 1 \u00b5g of the Bioss antibody against H3K4me3 (Cat. No. bs-53103R) as described above. The IP'd DNA was subsequently analyzed on an Illumina Genome Analyzer. Library preparation, cluster generation, and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. The figure shows the peak distribution along the complete sequence and a 1.2 Mb region of the X-chromosome (figure A and B) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure C and D). These results clearly show an enrichment of the H3K4 trimethylation at the promoters of active genes.

$ChIP assays were performed using human HeLa cells, the Bioss antibody against H3K4me3 (Cat. No. bs-53103R) and optimized PCR primer pairs for qPCR. ChIP was performed using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 1, 2, 5 and 10 \u00b5g of antibody per ChIP experiment was analyzed. IgG (2 \u00b5g\/IP) was used as a negative IP control. Figure A. Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as positive controls, and for the inactive MYOD1 gene, used as negative control. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that trimethylation of K4 at histone H3 is associated with the promoters of active genes Figure B. Recovery of the nucleosomes carrying the H3K4me1, H3K4me2, H3K4me3 modifications and the unmodified H3K4 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H3K4me3 modification. At higher concentrations, some H3K4me2 is also precipitated.$

ChIP assays were performed using human HeLa cells, the Bioss antibody against H3K4me3 (Cat. No. bs-53103R) and optimized PCR primer pairs for qPCR. ChIP was performed using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 1, 2, 5 and 10 \u00b5g of antibody per ChIP experiment was analyzed. IgG (2 \u00b5g\/IP) was used as a negative IP control. Figure A. Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as positive controls, and for the inactive MYOD1 gene, used as negative control. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that trimethylation of K4 at histone H3 is associated with the promoters of active genes Figure B. Recovery of the nucleosomes carrying the H3K4me1, H3K4me2, H3K4me3 modifications and the unmodified H3K4 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H3K4me3 modification. At higher concentrations, some H3K4me2 is also precipitated.

Applications

WB
IF
dot-blot
ChIP-seq

Reactivity

Human
Mouse
Others

Overview
Catalog #	bs-53103R
Product Name	H3K4me3 Polyclonal Antibody
Applications	WB, IF, dot-blot, ChIP-seq
Reactivity	Human, Mouse, Others
Specifications
Conjugation	Unconjugated
Host	Rabbit
Source	Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylated lysine 4 (H3K4me3), using a KLH-conjugated synthetic peptide
Clonality	Polyclonal
Isotype	Not Determined
Concentration	2ug/ul
Purification	Affinity purified polyclonal antibody in PBS containing 0.05% azide and 0.05% ProClin 300.
Storage Buffer	PBS with 0.05% sodium azide and 0.05% Proclin300.
Storage Condition	Store at -20°C for 12 months.
Target
Gene ID	8350
Swiss Prot	P68431
Background	Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling.
Application Dilution
WB	1:300-5000
IF
dot-blot
ChIP-seq