Anti-Histone H4 (acetyl K16) 抗体 [EPR1004] (ab109463)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 2 µg of ab109463 [EPR1004]. 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.

Additional screenshots of mapped reads can be downloaded here.

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

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.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

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 Fluorr^® 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. 

Intracellular 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). 

Intracellular 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).