H3K27me1 Polyclonal Antibody

$ChIP assays were performed using HeLa cells, the Bioss antibody against H3K27me1 (Cat. No. bs-53108R) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 100,000 cells. A titration of the antibody consisting of 1, 2, 5 and 10 \u03bcg per ChIP experiment was analyzed. IgG (2 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoter and the coding region of the active gene GAPDH used as a negative and a positive control target, respectively. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).$

ChIP assays were performed using HeLa cells, the Bioss antibody against H3K27me1 (Cat. No. bs-53108R) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 100,000 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 and the coding region of the active gene GAPDH used as a negative and a positive control target, respectively. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Bioss antibody directed against H3K27me1 (Cat. No. bs-53108R). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the purified antibody was estimated to be 1:32,900.

A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K27me1 (Cat. No. bs-53108R) with peptides containing other modifications and unmodified sequences of histone H3 and H4. 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 1:20,000. The figure shows a high specificity of the antibody for the modification of interest.

$Histone extracts (15 \u03bcg) from HeLa cells were analyzed by Western blot using the Bioss antibody against H3K27me1 (Cat. No. bs-53108R) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.$

Histone extracts (15 \u03bcg) from HeLa cells were analyzed by Western blot using the Bioss antibody against H3K27me1 (Cat. No. bs-53108R) 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.

$U2OS cells were stained with the Bioss antibody against H3K27me1 (Cat. No. bs-53108R) and with DAPI. Cells were fixed with 4% formaldehyde for 20\u2019 and blocked with PBS\/TX-100 containing 5% normal goat serum. Figure A: cells were immunofluorescently labeled with the H3K27me1 antibody (left) diluted 1:1,000 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right), which specifically labels DNA. Figure B, C, D, and E: staining of the cells with the H3K27me1 antibody after incubation of the antibody with 2 ng\/\u03bcl blocking peptide containing the unmodified and the mono-, di- and trimethylated H3K27, respectively.$

U2OS cells were stained with the Bioss antibody against H3K27me1 (Cat. No. bs-53108R) and with DAPI. Cells were fixed with 4% formaldehyde for 20\u2019 and blocked with PBS\/TX-100 containing 5% normal goat serum. Figure A: cells were immunofluorescently labeled with the H3K27me1 antibody (left) diluted 1:1,000 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right), which specifically labels DNA. Figure B, C, D, and E: staining of the cells with the H3K27me1 antibody after incubation of the antibody with 2 ng\/\u03bcl blocking peptide containing the unmodified and the mono-, di- and trimethylated H3K27, respectively.

Applications

WB
ELISA
IF
dot-blot

Reactivity

Human
Others

Overview
Catalog #	bs-53108R
Product Name	H3K27me1 Polyclonal Antibody
Applications	WB, ELISA, IF, dot-blot
Reactivity	Human, Others
Specifications
Conjugation	Unconjugated
Host	Rabbit
Source	Polyclonal antibody raised in rabbit against histone H3 containing the monomethylated lysine 27 (H3K27me1), 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
ELISA	1:500-1000
IF
dot-blot