Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (AB45166)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) treated (Red)/untreated (Green) with 500ng/ml Trichostatin A for 4 hours with purified ab45166 at 1/20 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (AB45166)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon tissue sections labelling Histone H4 (acetyl K8) with unpurified ab45166 at dilution of 1/200.

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (AB45166)

Immunohistochemical staining of paraffin-embedded human colon sections labelling Histone H4 (acetyl K8) with purified ab45166 at dilution of 1 : 2500. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• ChIP

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ChIP - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (AB45166)

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 2μg of ab45166 (red), and 20μl 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.

• IP

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Immunoprecipitation - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (AB45166)

ab45166 (purified) at 1/20 immunoprecipitating Histone H4 (acetyl K8) in HeLa treated with Trichostatin A whole cell lysate.

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

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (ab45166) at 1/20 dilution

Lane 1:

HeLa (human cervix adenocarcinoma) treated with Trichostatin A whole cell lysate at 10 µg

Lane 2:

(+) ab45166 + HeLa treated with Trichostatin A whole cell lysate

Lane 3:

(-) Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab45166 in HeLa treated with Trichostatin A whole cell lysate.

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/10000 dilution

Predicted band size: 11 kDa

Observed band size: 11 kDa

false

• ChIP-seq

Lab

ChIP-sequencing - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (AB45166)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 HeLa cells and 4 μg of ab45166 [EP1002Y]. 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (AB45166)

Immunohistochemical staining of paraffin-embedded rat kidney sections labelling Histone H4 (acetyl K8) with purified ab45166 at dilution of 1 : 2500. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (AB45166)

Immunocytochemistry/immunofluorescence staining of 4% paraformaldehyde fixed; 0.1% triton X 100 permeabilized C6 (rat glioma) cells (non-treated-top panels) and (C6 + TSA(500ng/ml, 4hr)-middle panels) with purified ab45166 at dilution of 1/150. The secondary antibody used was Alexa Fluor® 488; goat anti-rabbit IgG (ab150077) at a dilution of 1/1000. Nucleus was counter-stained with DAPI (blue). ab7291, a mouse anti-tubulin antibody (1/1000) was used to stain tubulin along with ab150120 (AlexaFluor®594 goat anti-mouse secondary, 1/1000) shown in the top right and middle right hand panels. The negative controls are shown in the bottom two panels- for negative control 1 rabbit primary antibody and anti-mouse secondary antibody (ab150120) was used. For negative control 2 mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077) was used.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (AB45166)

Immunohistochemical staining of paraffin-embedded mouse kidney sections labelling Histone H4 (acetyl K8) with purified ab45166 at dilution of 1 : 2500. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• WB

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Western blot - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (AB45166)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (ab45166) at 1/5000 dilution

Lane 1:

Untreated HeLa (human cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

HeLa (human cervix adenocarcinoma) treated with Trichostatin A whole cell lysate at 10 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: 11 kDa

Observed band size: 11 kDa

false

• WB

Unknown

Western blot - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (AB45166)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (ab45166) at 1/5000 dilution

Lane 1:

Untreated C6 (rat glioma) whole cell lysate at 10 µg

Lane 2:

C6 (rat glioma) treated with Trichostatin A whole cell lysate at 10 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: 11 kDa

Observed band size: 11 kDa

false

• WB

Unknown

Western blot - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (AB45166)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (ab45166) at 1/5000 dilution

Lane 1:

Untreated NIH/3T3 (mouse embryo) whole cell lysate at 10 µg

Lane 2:

NIH/3T3 (mouse embryo) treated with Trichostatin A whole cell lysate at 10 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: 11 kDa

Observed band size: 11 kDa

false