H4K5 8 12ac Polyclonal Antibody

$HeLa cells were stained with the Bioss antibody against H4K5,8,12ac (cat. No. bs-53096R) and with DAPI. Cells were fixed with 4% formaldehyde for 10\u2019 and blocked with PBS\/TX-100 containing 5% normal goat serum and 1% BSA. Figure A: cells were immunofluorescently labeled with the H4K5,8,12ac antibody (left) diluted 1:500 in blocking solution followed by Alexa488 conjugated secondary antibody. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. Figure B, C, D, and E: staining of the cells with the H4K5,8,12ac antibody after incubation of the antibody with 10 ng\/\u03bcl of the following blocking peptides: H4K5,8,12 unmodified (figure B), H4K5,8,12ac (figure C), H2A.ZK5,7,11ac (figure D) and H4K5,8,12,16ac (figure E).$

HeLa cells were stained with the Bioss antibody against H4K5,8,12ac (cat. No. bs-53096R) 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. Figure A: cells were immunofluorescently labeled with the H4K5,8,12ac antibody (left) diluted 1:500 in blocking solution followed by Alexa488 conjugated secondary antibody. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. Figure B, C, D, and E: staining of the cells with the H4K5,8,12ac antibody after incubation of the antibody with 10 ng\/\u03bcl of the following blocking peptides: H4K5,8,12 unmodified (figure B), H4K5,8,12ac (figure C), H2A.ZK5,7,11ac (figure D) and H4K5,8,12,16ac (figure E).

$Western blot was performed on whole cell (25 \u03bcg, lane 1) and histone extracts (15 \u03bcg, lane 2) of HeLa cells, and on 1 \u03bcg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Bioss antibody against H4K5,8,12ac (cat. No. bs-53096R). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk.$

Western blot was performed on whole cell (25 \u03bcg, lane 1) and histone extracts (15 \u03bcg, lane 2) of HeLa cells, and on 1 \u03bcg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Bioss antibody against H4K5,8,12ac (cat. No. bs-53096R). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk.

To test the cross-reactivity of the Bioss antibody against H4K5,8,12ac (cat. No. bs-53096R), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure shows a high specificity of the antibody for the modification of interest.

To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Bioss antibody directed against H4K5,8,12ac (cat. No. bs-53096R) 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 1:14,500.

$ChIP was performed with 0.5 \u03bcg of the Bioss antibody against H4K5,8,12ac (cat. No. bs-53096R) on sheared chromatin from 100,000 K562 cells using a ChIP-seq kit. The IP\u2019d DNA was subsequently analyzed on an Illumina Genome Analyzer. Library preparation, cluster generation, and sequencing were performed according to the manufacturer\u2019s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. The figure shows the signal distribution along the complete length of chromosome 2 (figure A) and a zoomin to a 600 kb region (figure B). Figure C and D show the enrichment in two genomic regions on chromosome 3 and 12, respectively, containing EIF4A2 and GAPDH positive controls.$

ChIP was performed with 0.5 \u03bcg of the Bioss antibody against H4K5,8,12ac (cat. No. bs-53096R) on sheared chromatin from 100,000 K562 cells using a ChIP-seq kit. The IP\u2019d DNA was subsequently analyzed on an Illumina Genome Analyzer. Library preparation, cluster generation, and sequencing were performed according to the manufacturer\u2019s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. The figure shows the signal distribution along the complete length of chromosome 2 (figure A) and a zoomin to a 600 kb region (figure B). Figure C and D show the enrichment in two genomic regions on chromosome 3 and 12, respectively, containing EIF4A2 and GAPDH positive controls.

$ChIP assays were performed using K562 cells, Bioss antibody against H4K5,8,12ac (cat. No. bs-53096R) and optimized PCR primer sets for qPCR. ChIP was performed with a ChIP- seq kit on sheared chromatin from 100,000 cells. A titration of the antibody consisting of 0.2, 0.5, 1 and 2 \u03bcg per ChIP experiment was analyzed. IgG (1 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active gene EIF4A2 and for a region 1 kb upstream of the GAPDH gene, used as positive controls, and for the inactive MYOD1 gene and the Sat2 satellite repeat region used as negative controls. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).$

ChIP assays were performed using K562 cells, Bioss antibody against H4K5,8,12ac (cat. No. bs-53096R) and optimized PCR primer sets for qPCR. ChIP was performed with a ChIP- seq kit on sheared chromatin from 100,000 cells. A titration of the antibody consisting of 0.2, 0.5, 1 and 2 \u03bcg per ChIP experiment was analyzed. IgG (1 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active gene EIF4A2 and for a region 1 kb upstream of the GAPDH gene, used as positive controls, and for the inactive MYOD1 gene and the Sat2 satellite repeat region used as negative controls. The 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
IP
IF
dot-blot
ChIP-seq

Reactivity

Human
Mouse

Overview
Catalog #	bs-53096R
Product Name	H4K5 8 12ac Polyclonal Antibody
Applications	WB, ELISA, IP, IF, dot-blot, ChIP-seq
Reactivity	Human, Mouse
Specifications
Conjugation	Unconjugated
Host	Rabbit
Source	Polyclonal antibody raised in rabbit against the region of histone H4 containing the acetylated lysines 5, 8 and 12 (H4K5,8,12ac), using a KLH-conjugated synthetic peptide
Clonality	Polyclonal
Isotype	Not Determined
Concentration	0.75ug/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	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
IP	1-2ug
IF
dot-blot
ChIP-seq