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.

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

Lane 1 : Untreated HeLa (human cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma) treated with Trichostatin A whole cell lysate

Lysates/proteins at 10 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

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 Fluor^® 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.

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.

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.

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

Lane 1 (input): HeLa treated with Trichostatin A whole cell lysate (10µg)

Lane 2 (+): ab45166 + HeLa treated with Trichostatin A whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab45166 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.

Lanes 1-2 : Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (ab45166) at 1/20 dilution
Lane 3 : Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) at 1/20 dilution

Lane 1 : HeLa (human cervix adenocarcinoma) treated with Trichostatin A whole cell lysate at 10 µg
Lanes 2-3 : HeLa (human cervix adenocarcinoma) treated with Trichostatin A whole cell lysate

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/10000 dilution

Observed band size: 11 kDa why is the actual band size different from the predicted?

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.

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

Lane 1 : Untreated C6 (rat glioma) whole cell lysate
Lane 2 : C6 (rat glioma) treated with Trichostatin A whole cell lysate

Lysates/proteins at 10 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

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.

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

Lane 1 : Untreated NIH/3T3 (mouse embryo) whole cell lysate
Lane 2 : NIH/3T3 (mouse embryo) treated with Trichostatin A whole cell lysate

Lysates/proteins at 10 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

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.