Recombinant Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (ab32129)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ HeLa cells and 4 µg of Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (ab32129). ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

Additional screenshots of mapped reads can be downloaded here.

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 30 µg of chromatin and 4 µg of Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (ab32129). ChIP DNA was sequenced on the Illumina NextSeq 500 to a depth of 30 million reads. ChIP-Seq validation performed by Active Motif, Carlsbad, CA.

Additional screenshots of mapped reads can be downloaded here.

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. 
The ChIP was performed with 25 µg of chromatin, 2 µg of ab32129 (red), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). 
Primers and probes are located in the first kb of the transcribed region.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Histone H3 with purified ab32129 at 1/250 dilution (4 µg/mL). Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL) was used as the secondary antibody only control.

IHC image of ab32129 staining Histone H3 (acetyl K9) 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 ab32129, 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.

ab32129 (purified) at 1/30 dilution (20 µg/ml) immunoprecipitating Histone H3 acetyl K9 in TSA treated HeLa whole cell lysate.
Lane 1 (input): HeLa (human cervix adenocarcinoma epithelial cell) treated with 500ng/ml TSA for 4h whole cell lysate 10µg
Lane 2 (+): ab32129 & TSA treated HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32129 in TSA treated HeLa whole cell lysate
For western blotting, ab32129 at 1/500 and veriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM /TBST.

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) treated (Red)/untreated (Green) with 500ng/ml Trichostatin A for 4 hours with purified ab32129 at 1/230 dilution. The secondary antibody was Goat anti rabbit IgG (Alexa Fluorr^® 488) at 1/2000 dilution. A Rabbit monoclonal IgG (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control. 

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using ab32129 at 1/50 - 1/100

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

CUT&Tag-seq was performed using 200,000 Oli-neu (Oligodendrocyte progenitor) cells.  Cells were permeabilized with 0.05% Digitonin and 0.01% NP-40 for 3 minutes.  A 1:100 dilution of Recombinant Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (ab32129) was used, along with a Guinea pig anti-rabbit Secondary.  DNA was seg using Illumina NovaSeq S Prime to a depth of 24 million reads.

This image is courtesy of Dr Marek Bartosovic, Gonçalo Castelo-Branco Group, Karolinska Institutet.