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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

Immunohistochemical analysis of Paraffin-embedded sections human colorectal carcinoma tissue labelling Histone H3 (acetyl K9) with ab32129 at 1/2000 dilution, followed by a ready to use secondary Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Staining on human colorectal carcinoma tissue is observed. Counter stained with Haematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

Immunohistochemical analysis of Paraffin-embedded sections human cerebrum tissue labelling Histone H3 (acetyl K9) with ab32129 at 1/2000 dilution, followed by a ready to use secondary Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Staining on human cerebrum tissue is observed. Counter stained with Haematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

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.

• ChIP-seq

Lab

ChIP-sequencing - 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.

• ChIP

Unknown

ChIP - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

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.

• ChIP-seq

Supplier Data

ChIP-sequencing - 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 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.

• IP

Lab

Immunoprecipitation - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

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.

All lanes:

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

Predicted band size: 15 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

Immunohistochemical analysis of Paraffin-embedded sections mouse colon tissue labelling Histone H3 (acetyl K9) with ab32129 at 1/6000 dilution, followed by a ready to use secondary Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Staining on mouse colon tissue is observed. Counter stained with Haematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

Immunohistochemical analysis of Paraffin-embedded sections rat colon tissue labelling Histone H3 (acetyl K9) with ab32129 at 1/6000 dilution, followed by a ready to use secondary Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Staining on rat colon tissue is observed. Counter stained with Haematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• WB

Lab

Western blot - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. ab181602 was used as GAPDH loading control. ab176842 was for Histone H3 detection.

All lanes:

Western blot - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (ab32129) at 1/10000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) treated with 500ng/ml Trichostatin A for 4 hours whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 15 kDa

Observed band size: 15 kDa

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• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human epithelial cell line from cervix adenocarcinoma) cells and 5 µg of ab32129 [Y28]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human epithelial cell line from cervix adenocarcinoma) cells and 5 µg of ab32129 [Y28]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human epithelial cell line from cervix adenocarcinoma) cells and 5 µg of ab32129 [Y28]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Lab

Western blot - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. ab181602 was used as GAPDH loading control. ab176842 was for Histone H3 detection.

All lanes:

Western blot - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (ab32129) at 1/10000 dilution

Lane 1:

C6 (Rat glial tumor glial cell) whole cell lysate at 15 µg

Lane 2:

C6 (Rat glial tumor glial cell) treated with 500ng/ml Trichostatin A for 4 hours whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 15 kDa

Observed band size: 15 kDa

false

• WB

Lab

Western blot - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. ab181602 was used as GAPDH loading control. ab176842 was for Histone H3 detection.

All lanes:

Western blot - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (ab32129) at 1/10000 dilution

Lane 1:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 15 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast) treated with 500ng/ml Trichostatin A for 4 hours whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 15 kDa

Observed band size: 15 kDa

false

• CUT&Tag-seq

Supplier Data

CUT&Tag sequencing - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

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.

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

• CUT&Tag

Supplier Data

CUT&Tag - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

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. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

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

• PepArr

Lab

Peptide Array - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (AB32129)

ab32129 was tested in Peptide array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
Circle area represents affinity between the antibody and a peptide : all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.

The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, is available under the Product Protocol section.

Please note this antibody has weak cross-reactivity with H3R8me1 and H3K4me3 in peptide array. Further optimization may be needed to minimize the cross-reactivity in assays similar to peptide array.