Anti-Histone H2B (crotonyl K5) antibody [EPR17483] (ab177396) at 1/1000 dilution + HeLa (Human epithelial cells from cervix adenocarcinoma) cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 14 kDa
Observed band size: 14 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Histone H2B (crotonyl K5) antibody [EPR17483] (ab177396) at 1/500 dilution + NIH/3T3 (Mouse embyro fibroblast cells) cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 14 kDa
Observed band size: 14 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

ab177396 was tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.
The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H2B (crotonyl K5) using ab177396 at 1/500 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Counter stained with Hematoxylin.
Inset image: negative control obtained using PBS instead of ab177396.
Note: Nucleus staining on glandular epithelium of Human colon tissue was observed.

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

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling Histone H2B (crotonyl K5) using ab177396 at 1/500 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Counter stained with Hematoxylin.
Inset image: negative control obtained using PBS instead of ab177396.
Note: Nucleus staining on glandular epithelium of Mouse colon tissue was observed.

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

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling Histone H2B (crotonyl K5) using ab177396 at 1/500 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Counter stained with Hematoxylin.
Inset image: negative control obtained using PBS instead of ab177396.
Note: Nucleus staining on Rat liver tissue was observed.

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H2B (crotonyl K5) with ab177396 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
1. ab177396 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.