All lanes : Anti-Histone H1.0 antibody [34] (ab11079) 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 - ab11079 observed at 30 kDa. Red - loading control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55 kDa.

ab11079 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 ab11079 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 H1.0 staining in a formalin fixed, paraffin embedded human breast adenocarcinama 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 ab11079, 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.

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

All lanes : Anti-Histone H1.0 antibody [34] (ab11079) at 1 µg/ml

Lane 1 : Histone H1.0 (Human) - Recombinant Protein
Lane 2 : Histone H1.2 (Human) - Recombinant Protein

Lysates/proteins at 0.1 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) (ab65485) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 20 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

Anti-Histone H1.0 antibody [34] (ab11079) at 1 µg/ml + HeLa (Triton-enriched) Cell Lysate at 10 µg

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) (ab65485) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 20 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?
Additional bands at: 120 kDa, 18 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 8 minutes

Western blot using ab11079 on HeLa whole cell extract (30 µg/lane).

Exposure 5 sec using ECL.
12.5% SDS-PAGE gel.

Mouse monoclonal to Histone H1.0 against recombinant human H1 isoforms H1.0 to H1.5 as depicted in the figure.

Dilution 1/1000.

Recombinant H1 isoforms courtesy of Dr Nicole Happel.

(see attached review)