Anti-Histone H2A (mono methyl R3) + Histone H4 (mono methyl R3) antibody [EPR16995] (ab177186) at 1/1000 dilution + HeLa (human epithelial cells from cervix adenocarcinoma) cell lysate at 10 µg

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

Predicted band size: 11, 14 kDa
Observed band size: 11 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Histone H2A (mono methyl R3) + Histone H4 (mono methyl R3) antibody [EPR16995] (ab177186) at 1/1000 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: 11, 14 kDa
Observed band size: 11 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

ab177186 was tested in Peptide Array against human synthetic peptides to different Histone modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to histone H2A R3me1peptide (ab28756) and H4 R3me1peptide(ab17770), indicating that this antibody specifically recognises the histone H2A R3me1peptide (ab28756) and H4 R3me1peptide(ab17770).

ab28756 - H2A R3me1
ab17770 - H4 R3me1
ab178994 - H2A unmod(1-30)
ab179297 - H4 unmod(1-30)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H2A (mono methyl R3) + Histone H4 (mono methyl R3) with ab177186 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus staining on Human colon tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling Histone H2A (mono methyl R3) + Histone H4 (mono methyl R3) with ab177186 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus staining on mouse colon tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling Histone H2A (mono methyl R3) + Histone H4 (mono methyl R3) with ab177186 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus staining on rat colon tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 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 H2A (mono methyl R3) + Histone H4 (mono methyl R3) with ab177186 at 1/2000 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:
-ve control 1: ab177186 at 1/4000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 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.