Lanes 1-2 : Anti-Histone H4 (acetyl K16) antibody [EPR1004] (ab109463) at 1/6000 dilution
Lanes 3-4 : Anti-Histone H4 (acetyl K16) antibody [EPR1004] (ab109463) at 1/24000 dilution

Lanes 1 & 3 : Untreated C6 (Rat glial tumor glial cell) whole cell lysate
Lanes 2 & 4 : C6 (Rat glial tumor glial cell) treated with Trichostatin A (final concentration is 500ng/ml) for 4 hours whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 11 kDa


Exposure time: 3 minutes

Blocking/Diluting buffer and concentration 5% NFDM/TBST

Immunocytochemistry/ Immunofluorescence analysis of untreated HeLa cells (top row) and HeLa+ TSA(500ng/ml, 4h) cells (middle row) labeling Histone H4 (acetyl K16) with ab109463 at 1/500. Goat anti rabbit IgG(Alexa Fluor^® 488); ab150077 at 1/1000 dilution was used as the secondary antibody. Cells were fixed with 4% paraformaldehyde and permeabilised with 0.1% tritonX-100. DAPI (blue) was used as a nuclear counterstain.

All lanes : Anti-Histone H4 (acetyl K16) antibody [EPR1004] (ab109463) at 1/1000 dilution (unpurified)

Lane 1 : HeLa cell lysates, untreated
Lane 2 : HeLa cell lysates treated with TSA

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP-labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 11 kDa
Observed band size: 11 kDa

Anti-Histone H4 (acetyl K16) antibody [EPR1004] (ab109463) at 1/1000 dilution (unpurified) + HeLa cell lysate - treated with TSA at 10 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 11 kDa
Observed band size: 11 kDa

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-Histone H4 (acetyl K16) antibody [EPR1004] (ab109463) at 1/1500 dilution (purified) + HeLa cell lysate - treated with TSA at 10 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 11 kDa
Observed band size: 11 kDa

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-Histone H4 (acetyl K16) antibody [EPR1004] (ab109463) at 1/1000 dilution (unpurified) + C6 cell lysate - treated with TSA at 10 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 11 kDa
Observed band size: 11 kDa

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-Histone H4 (acetyl K16) antibody [EPR1004] (ab109463) at 1/1500 dilution (unpurified) + C6 cell lysate - treated with TSA at 10 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 11 kDa
Observed band size: 11 kDa

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-Histone H4 (acetyl K16) antibody [EPR1004] (ab109463) at 1/1000 dilution (purified) + Mouse spleen tissue lysate at 10 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L)

Predicted band size: 11 kDa
Observed band size: 11 kDa

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-Histone H4 (acetyl K16) antibody [EPR1004] (ab109463) at 1/1500 dilution (purified) + Mouse spleen tissue lysate at 10 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 11 kDa
Observed band size: 11 kDa

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue labelling Histone H4 (acetyl K16) with unpurified ab109463 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue labelling Histone H4 (acetyl K16) with purified ab109463 at 1/150. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue labelling Histone H4 with unpurified ab109463 at 1/100.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human transitional cell carcinoma labelling Histone H4 (acetly K16) with unpurified ab109463 at 1/100.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Histone H4 (acetyl K16) with unpurified ab109463 (red) at 1/100. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/100) and secondary antibody ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Immunocytochemistry/Immunofluorescence analysis of HeLa cells treated with TSA labelling Histone H4 (acetyl K16) with unpurified ab109463 (red) at 1/100. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/100) and secondary antibody ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Histone H4 (acetyl K16) with purified ab109463 (red) at 1/150. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/150) and secondary antibody ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Immunocytochemistry/Immunofluorescence analysis of HeLa cells treated with TSA labelling Histone H4 (acetyl K16) with purified ab109463 (red) at 1/150. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/150) and secondary antibody ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Overlay histogram showing HeLa cells stained with unpurified ab109463 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab109463, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Flow cytometry analysis of HeLa cells labelling Histone H4 (acetyl K16) with unpurified ab109463 (red) at 1/130. Cells were fixed with 80% methanol. A FITC-conjugated goat anti-rabbit IgG was used as the secondary antibody (1/150). A rabbit monoclonal IgG was used as the isotype control (green).

Flow cytometry analysis of HeLa cells labelling Histone H4 (acetyl K16) with purified ab109463 (red) at 1/200. Cells were fixed with 80% methanol. A FITC-conjugated goat anti-rabbit IgG was used as the secondary antibody (1/150). A rabbit monoclonal IgG was used as the isotype control (green).