Chromatin was prepared from MEF (Mouse embryonic fibroblast cell line) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab51997 (red), and 20 μl protein A/G sepharose beads. 2μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

All lanes : Anti-Histone H4 (acetyl K5) antibody [EP1000Y] - ChIP Grade (ab51997) at 1/500000 dilution (purified)

Lane 1 : Nuclear extract of HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 7mM Sodium Butyrate for 24 hours
Lane 2 : Untreated nuclear extract of HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast cell line) treated with 500ng/ml Trichostatin A for 4 hours whole cell lysates
Lane 4 : Untreated NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysates
Lane 5 : C6 (Rat glial tumor cell line) treated with 500ng/ml Trichostatin A for 4 hours whole cell lysates
Lane 6 : Untreated C6 (Rat glial tumor cell line) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 11 kDa
Observed band size: 11 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

IHC image of unpurified ab51997 staining Histone H4 (acetyl K5) in human colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab51997, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma)treated with 500ng/m Trichostatin A for 4 hours labeling Histone H4 (acetyl K5) with purified ab51997 at 1/5000 dilution (0.1μg/ml). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. ab195889, an Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain at 1/200 (2.5 μg/ml). ab150077, a Goat anti rabbit IgG(Alexa Fluor^® 488) secondary antibody was used at 1/1000 dilution. PBS instead of the primary antibody was used as a control. DAPI nuclear staining.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue sections labeling Histone H4 (acetyl K5) with purified ab51997 at 1/500 dilution (1 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, PH9. Hematoxylin was used to counter stain. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. PBS instead of the primary antibody was used as the negative control.

Direct ELISA with Histone H4 K5ac peptide, Histone H4 K8ac peptide, Histone H4 K16me1 peptide, Histone H4 K16me3 peptide, and Histone H4 unmodified peptide, all at 10ng/ml. ab51997 used as the primary antibody at a range of 0~1000ng/ml. Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) used as the secondary antibody at 1:2500 dilution.

Direct ELISA with Histone H4 K5ac peptide, Histone H4 K8ac peptide, Histone H4 K16me1 peptide, Histone H4 K16me3 peptide, and Histone H4 unmodified peptide, all at 100ng/ml. ab51997 used as the primary antibody at a range of 0~1000ng/ml. Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) used as the secondary antibody at 1:2500 dilution.

Direct ELISA with Histone H4 K5ac peptide, Histone H4 K8ac peptide, Histone H4 K16me1 peptide, Histone H4 K16me3 peptide, and Histone H4 unmodified peptide, all at 1000ng/ml. ab51997 used as the primary antibody at a range of 0~1000ng/ml. Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) used as the secondary antibody at 1:2500 dilution.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebral cortex tissue sections labeling Histone H4 (acetyl K5) with purified ab51997 at 1/500 dilution (1 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, PH9. Hematoxylin was used to counter stain. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue sections labeling Histone H4 (acetyl K5) with purified ab51997 at 1/500 dilution (1 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, PH9. Hematoxylin was used to counter stain. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-Histone H4 (acetyl K5) antibody [EP1000Y] - ChIP Grade (ab51997) at 1/1000000 dilution (unpurified)

Lane 1 : Untreated HeLa cells
Lane 2 : TSA treated HeLa calls

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP labelled (1:2000)

Predicted band size: 11 kDa
Observed band size: 11 kDa

ab51997 (purified) at 1/30 dilution (2µg) immunoprecipitating Histone H4 (acetyl K5) in HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with TSA whole cell lysate.

Lane 1 (input): HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with TSA whole cell lysate 10ug
Lane 2 (+): ab51997+ HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with TSA whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab51997 in HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with TSA whole cell lysate

For western blotting, ab131366 VeriBlot for IP (HRP) was used for detection (1/10000).

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

ICC/IF image of unpurtified ab51997 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab51997, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.