All lanes : Anti-Histone H1.0 antibody [27] (ab11080) at 1/500 dilution

Lane 1 : Wild-type A431 cell lysate
Lane 2 : H1F0 knockout A431 cell lysate
Lane 3 : MCF7 cell lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Predicted band size: 20 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?

Lanes 1 - 3: Merged signal (red and green). Green - ab11080 observed at 30 kDa. Red - loading control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55 kDa.

ab11080 was shown to react with Histone H1.0 in wild-type A431 cells in Western blot with loss of signal observed in H1F0 knockout sample. Wild-type A431 and H1F0 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab11080 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4°C at a 1 in 500 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

IHC image of Histone H1 staining in a section of formalin-fixed paraffin-embedded [human normal colon]*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was then incubated with ab11080, 1/1000 dilution, for 15 mins at room temperature. A goat anti-mouse biotinylated secondary antibody (ab6788, 1/1000 dilution), was used to detect the primary, and visualized using an HRP conjugated ABC system. Streptavidin HRP was used, ab7403 at a 1/10000 dilution. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

All lanes : Anti-Histone H1.0 antibody [27] (ab11080) at 1/500 dilution

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lane 3 : Histone H1.0 Human Recombinant Protein

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 20 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?
Additional bands at: 46 kDa, 65 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 1 minute

IHC image of Histone H1.0 staining in human colon formalin fixed paraffin embedded tissue section*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab11080, 7.5µg/ml overnight at +4°C. An HRP-conjugated secondary (ab97240, 1/2000 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

The inset negative control image is secondary-only at 1/500 dilution.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

IHC image of Histone H1.0 staining in Human pancreas adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab11080, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

 

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.

Overlay histogram showing HeLa cells stained with ab11080 (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 (ab11080, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.