H4K20me1 Polyclonal Antibody

$HeLa cells were stained with the Bioss antibody against H4K20me1 (Cat. bs-53104R) and with DAPI. Cells were fixed with 4% formaldehyde for 10\u2019 and blocked with PBS\/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me1 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.$

HeLa cells were stained with the Bioss antibody against H4K20me1 (Cat. bs-53104R) 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 H4K20me1 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.

$Western blot was performed on whole cell extracts (25 \u03bcg, lane 1) from HeLa cells, and on 1 \u03bcg of recombinant histone H2A, H2B, H3 and H4 (lane 2, 3, 4 and 5, respectively) using the Bioss antibody against H4K20me1 (cat. No. bs-53104R). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left, the position of the protein is indicated on the right.$

Western blot was performed on whole cell extracts (25 \u03bcg, lane 1) from HeLa cells, and on 1 \u03bcg of recombinant histone H2A, H2B, H3 and H4 (lane 2, 3, 4 and 5, respectively) using the Bioss antibody against H4K20me1 (cat. No. bs-53104R). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left, the position of the protein is indicated on the right.

To check the specificity of the Bioss antibody against H4K20me1 (cat. No bs-53104R) a Dot Blot was performed with peptides containing different modifications of histone H3 and H4 or the unmodified H4K20 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 1:20,000. The figure shows a high specificity of the antibody for the modification of interest.

To determine the titer, an ELISA was performed using a serial dilution of the Bioss antibody directed against H4K20me1 (cat. No. bs-53104R) in antigen-coated wells. 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 1:51,100.

$ChIP assays were performed using HeLa cells, the Bioss antibody against H4K20me1 (cat. No. bs-53104R) and optimized PCR primer sets for qPCR. ChIP was performed with a ChIP-seq kit on sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5, and 10 \u03bcg per ChIP experiment was analyzed. IgG (2 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the coding region of the active GAPDH and ACTB genes, used as positive controls, and for the GAPDH promoter and a region located 1 kb u pstream of the GAPDH promoter, used as negative controls. Figureshows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).$

ChIP assays were performed using HeLa cells, the Bioss antibody against H4K20me1 (cat. No. bs-53104R) and optimized PCR primer sets for qPCR. ChIP was performed with a ChIP-seq kit on 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 coding region of the active GAPDH and ACTB genes, used as positive controls, and for the GAPDH promoter and a region located 1 kb u pstream of the GAPDH promoter, used as negative controls. Figureshows 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)
dot-blot

Reactivity

Human

Overview
Catalog #	bs-53104R
Product Name	H4K20me1 Polyclonal Antibody
Applications	WB, ELISA, IF(ICC), dot-blot
Reactivity	Human
Specifications
Conjugation	Unconjugated
Host	Rabbit
Source	Polyclonal antibody raised in rabbit against histone H4 containing the monomethylated lysine 20 (H4K20me1), using a KLH-conjugated synthetic peptide
Clonality	Polyclonal
Isotype	Not Determined
Concentration	2ug/ul
Purification	Protein A 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	121504
Swiss Prot	P62805
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
dot-blot