H3K9me2 (9B3) Monoclonal Antibody

$Histone extracts (15 \u03bcg) from HeLa cells were analysed by Western blot using H3K9me2 (9B3) Monoclonal Antibody (Cat. No. bsm-53016M) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.$

Histone extracts (15 \u03bcg) from HeLa cells were analysed by Western blot using H3K9me2 (9B3) Monoclonal Antibody (Cat. No. bsm-53016M) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

$HeLa cells were stained with H3K9me2 (9B3) Monoclonal Antibody (bsm-53016M). Cells were fixed with 4% formaldehyde for 10\u2019 and blocked with PBS\/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K9me2 antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.$

HeLa cells were stained with H3K9me2 (9B3) Monoclonal Antibody (bsm-53016M). 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 labelled with the H3K9me2 antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

To test the specificity an ELISA was performed using a serial dilution H3K9me2 (9B3) Monoclonal Antibody (bsm-53016M). The wells were coated with peptides containing the unmodified H3K9 as well as the mono-, di- and trimethylated H3K9 and the dimethylated H3K4 and H3K27. Figure shows a high specificity of the antibody for the modification of interest.

$ChIP assays were performed using HeLa cells, H3K9me2 (9B3) Monoclonal Antibody (bsm-53016M) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 1.6 million cells. A titration of the antibody consisting of 1, 3 and 9 \u03bcg per ChIP experiment was analysed. IgG (5 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoter and the coding region of the GAPDH gene, and for the RPL10 and HBB promoters. Figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).$

ChIP assays were performed using HeLa cells, H3K9me2 (9B3) Monoclonal Antibody (bsm-53016M) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 1.6 million cells. A titration of the antibody consisting of 1, 3 and 9 \u03bcg per ChIP experiment was analysed. IgG (5 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoter and the coding region of the GAPDH gene, and for the RPL10 and HBB promoters. Figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Applications

WB
ELISA
IF(ICC)
IF

Reactivity

Human

Overview
Catalog #	bsm-53016M
Product Name	H3K9me2 (9B3) Monoclonal Antibody
Applications	WB, ELISA, IF(ICC), IF
Reactivity	Human
Specifications
Conjugation	Unconjugated
Host	Mouse
Source	Monoclonal antibody raised in mouse against histone H3, dimethylated at lysine 9 (H3K9me2), using a KLH-conjugated synthetic peptide
Clonality	Monoclonal
Clone #	9B3
Isotype	IgG1
Concentration	1ug/ul
Purification	Protein A purified monoclonal antibody in PBS containing 0.05% azide.
Storage Buffer	PBS with 0.05% sodium azide.
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
ELISA	1:500-1000
IF(ICC)	1:50-200
IF