Anti-Histone H4 (acetyl K8) antibody - ChIP Grade

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (acetyl K8) antibody - ChIP Grade (AB15823)

IHC image of histone H4 (acetyl K8) antibody staining in a section of formalin-fixed paraffin-embedded human breast adenocarcinoma* performed on a Leica BOND^TM system using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab15823, 0.5ug/ml, 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.

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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H4 (acetyl K8) antibody - ChIP Grade (AB15823)

ab15823 staining Histone H4 (Acetyl K8) in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab15823 at 0.1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ChIP

Unknown

ChIP - Anti-Histone H4 (acetyl K8) antibody - ChIP Grade (AB15823)

Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 μg of chromatin, 2 μg of ab15823 (blue), and 20 μl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

• WB

Unknown

Western blot - Anti-Histone H4 (acetyl K8) antibody - ChIP Grade (AB15823)

The blot shows that a H4 peptide containing an acetylated K8 residue was able to block recognition of histones by ab15823. Peptides containing other acetylated K residues of H4 and H2A did not block binding of histones by ab15823. This suggests ab15823 specifically recognises Histone H4 acetyl K8.

All lanes:

Western blot - Anti-Histone H4 (acetyl K8) antibody - ChIP Grade (ab15823) at 1 µg/mL

Lane 1:

Sodium butyrate treated hela histone at 2.5 µg

Lane 2:

Untreated hela histone at 2.5 µg

Lane 3:

Sodium butyrate treated hela histone at 2.5 µg with Human Histone H4 (acetyl K8) peptide (<a href='/en-us/products/unavailable/human-histone-h4-acetyl-k8-peptide-ab15824'>ab15824</a>)

Lane 4:

Untreated hela histone at 2.5 µg with Human Histone H4 (acetyl K8) peptide (<a href='/en-us/products/unavailable/human-histone-h4-acetyl-k8-peptide-ab15824'>ab15824</a>)

Lane 5:

Sodium butyrate treated hela histone at 2.5 µg with Human Histone H4 peptide (<a href='/en-us/products/unavailable/human-histone-h4-peptide-ab15825'>ab15825</a>)

Lane 6:

Untreated hela histone at 2.5 µg with Human Histone H4 peptide (<a href='/en-us/products/unavailable/human-histone-h4-peptide-ab15825'>ab15825</a>)

Lane 7:

Sodium butyrate treated hela histone at 2.5 µg with Histone H4 peptide - acetyl K5 (<a href='/en-us/products/unavailable/histone-h4-peptide-acetyl-k5-ab15596'>ab15596</a>)

Lane 8:

Untreated hela histone at 2.5 µg with Histone H4 peptide - acetyl K5 (<a href='/en-us/products/unavailable/histone-h4-peptide-acetyl-k5-ab15596'>ab15596</a>)

Lane 9:

Sodium butyrate treated hela histone at 2.5 µg with Human Histone H4 (acetyl K12) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h4-acetyl-k12-peptide-ab15662'>ab15662</a>)

Lane 10:

Untreated hela histone at 2.5 µg with Human Histone H4 (acetyl K12) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h4-acetyl-k12-peptide-ab15662'>ab15662</a>)

Lane 11:

Sodium butyrate treated hela histone at 2.5 µg with Histone H2A peptide - acetyl K9

Lane 12:

Untreated hela histone at 2.5 µg with Histone H2A peptide - acetyl K9

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 11 kDa

Observed band size: 12 kDa,22 kDa

false

• WB

Project

Western blot - Anti-Histone H4 (acetyl K8) antibody - ChIP Grade (AB15823)

All lanes:

Western blot - Anti-Histone H4 (acetyl K8) antibody - ChIP Grade (ab15823) at 1 µg/mL

Lane 1:

Calf Thymus Histone Preparation Nuclear Lysate (<a href='/en-us/products/unavailable/calf-thymus-histone-preparation-nuclear-lysate-ab121'>ab121</a>) at 0.5 µg/mL

Lane 2:

Calf Thymus Histone Preparation Nuclear Lysate (<a href='/en-us/products/unavailable/calf-thymus-histone-preparation-nuclear-lysate-ab121'>ab121</a>) at 0.5 µg/mL with Human Histone H4 peptide (<a href='/en-us/products/unavailable/human-histone-h4-peptide-ab15825'>ab15825</a>)

Lane 3:

Calf Thymus Histone Preparation Nuclear Lysate (<a href='/en-us/products/unavailable/calf-thymus-histone-preparation-nuclear-lysate-ab121'>ab121</a>) at 0.5 µg/mL with Human Histone H4 (acetyl K8) peptide (<a href='/en-us/products/unavailable/human-histone-h4-acetyl-k8-peptide-ab15824'>ab15824</a>)

Lane 4:

Calf Thymus Histone Preparation Nuclear Lysate (<a href='/en-us/products/unavailable/calf-thymus-histone-preparation-nuclear-lysate-ab121'>ab121</a>) at 0.5 µg/mL with Histone H4 peptide - acetyl K5

Lane 5:

Calf Thymus Histone Preparation Nuclear Lysate (<a href='/en-us/products/unavailable/calf-thymus-histone-preparation-nuclear-lysate-ab121'>ab121</a>) at 0.5 µg/mL with Human Histone H4 (acetyl K12) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h4-acetyl-k12-peptide-ab15662'>ab15662</a>)

Lane 6:

Calf Thymus Histone Preparation Nuclear Lysate (<a href='/en-us/products/unavailable/calf-thymus-histone-preparation-nuclear-lysate-ab121'>ab121</a>) at 0.5 µg/mL with Human Histone H2A (acetyl K9) peptide (<a href='/en-us/products/unavailable/human-histone-h2a-acetyl-k9-peptide-ab54016'>ab54016</a>)

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 11 kDa

Observed band size: 13 kDa

false

Exposure time: 3min