H3K27me2 Polyclonal Antibody

$ChIP assays were performed using HeLa cells, the Bioss antibody against H3K27me2 (cat. No. bs-53109R) and optimized PCR primer sets for qPCR. ChIP was performed with an Auto Histone ChIP-seq kit on the SX-8G IP-Star Compact automated system, using sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5, and 10 \u03bcg per ChIP experiment was analyzed. IgG (2 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the promoter of the inactive HBB and the coding region of the inactive MYOD1 genes, used as positive controls. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that H3K27me2 is preferably present at silent genes.$

ChIP assays were performed using HeLa cells, the Bioss antibody against H3K27me2 (cat. No. bs-53109R) and optimized PCR primer sets for qPCR. ChIP was performed with an Auto Histone ChIP-seq kit on the SX-8G IP-Star Compact automated system, using sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5, and 10 \u03bcg per ChIP experiment was analyzed. IgG (2 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the promoter of the inactive HBB and the coding region of the inactive MYOD1 genes, used as positive controls. The figure 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 H3K27me2 is preferably present at silent genes.

To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Bioss antibody directed against H3K27me2 (cat. No. bs-53109R). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:480,000.

A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K27me2 (cat. No. bs-53109R) with peptides containing other modifications of histone H3 and H4 and the unmodified sequence. One hundred to 0.2 pmol of the peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:50,000. The figure shows a high specificity of the antibody for the modification of interest.

$Histone extracts (15 \u03bcg) from HeLa cells were analyzed by Western blot using the Bioss antibody against H3K27me2 (cat. No. bs-53109R) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.$

Histone extracts (15 \u03bcg) from HeLa cells were analyzed by Western blot using the Bioss antibody against H3K27me2 (cat. No. bs-53109R) 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.

$HeLa cells were stained with the Bioss antibody against H3K27me2 (cat. No. bs-53109R) and with DAPI. Cells were fixed with 4% formaldehyde for 10\u2019 and blocked with PBS\/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H3K27me2 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.$

HeLa cells were stained with the Bioss antibody against H3K27me2 (cat. No. bs-53109R) 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 H3K27me2 antibody (left) diluted 1:500 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.

Applications

WB
ELISA
IF
dot-blot

Reactivity

Human

Overview
Catalog #	bs-53109R
Product Name	H3K27me2 Polyclonal Antibody
Applications	WB, ELISA, IF, dot-blot
Reactivity	Human
Specifications
Conjugation	Unconjugated
Host	Rabbit
Source	Polyclonal antibody raised in rabbis against the histone H3, dimethylated at lysine 27 (H3K27me2), using a KLH-conjugated synthetic peptide
Clonality	Polyclonal
Isotype	Not Determined
Concentration	2.5ug/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
ELISA	1:500-1000
IF
dot-blot