ChIP was performed with 0.5 µg ab233193 on sheared chromatin from 100,000 K562 cells. The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s 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.

All lanes : Anti-Histone H4 (acetyl K5 + K8 + K12) antibody (ab233193) at 1/1000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell extract at 25 µg
Lane 2 : HeLa histone extract at 15 µg
Lane 3 : Recombinant histone H2A at 1 µg
Lane 4 : Recombinant histone H2B at 1 µg
Lane 5 : Recombinant histone H3 at 1 µg
Lane 6 : Recombinant histone H4 at 1 µg

Dilution buffer: TBS-Tween containing 5% skimmed milk.

ChIP assays were performed using human K562 (Human chronic myelogenous leukemia cell line from bone marrow) cells, ab233193 and optimized PCR primer sets for qPCR. ChIP was performed on sheared chromatin from 100,000 cells. A titration of the antibody consisting of 0.2, 0.5, 1 and 2 µg per ChIP experiment was analysed. IgG (1 µg/IP) was used as negative IP control. QPCR was performed with primers for 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. Image shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were stained with ab233193 and with DAPI.

Cells were fxed with 4% formaldehyde for 10 minutes and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA.

Figure A: Cells were immunofluorescently labeled with ab233193 (left) diluted 1/500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa^®488. 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 ab233193 after incubation of the antibody with 10 ng/µl of the following blocking peptides: H4K5,8,12 unmodifed (B), H4K5,8,12ac (C), H2A.ZK5,7,11ac (D) and H4K5,8,12,16ac (E).

To test the cross reactivity of ab233193 a Dot Blot analysis was performed with peptides containing other histone modifcations and the unmodifed H4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. ab233193 was used at a dilution of 1:20,000.