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 (blue), and 20µl 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45166).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45166).

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, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45166).