All lanes : Anti-Histone H1.0 antibody [EPR6537] (ab125027) at 1/1000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 21 kDa
Observed band size: 33 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab125027 observed at 33 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab125027 was shown to react with H1F0 in A431 wild-type cells in Western blot. Loss of signal was observed when H1F0 knockout sample was used. A431 wild-type 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 ab125027 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Histone H1.0 antibody [EPR6537] (ab125027) at 10 µg (purified)

Lane 1 : human fetal lung lysate
Lane 2 : BxPC-3 lysate
Lane 3 : A431 lysate
Lane 4 : human fetal kidney lysate

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 21 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Immunofluorescence staining of BxPC-3 cells with purified ab125027 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 100% methanol and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab125027 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

Immunohistochemical staining of paraffin embedded human thyroid carcinoma with purified ab125027 at a working dilution of 1/400. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin embedded human pancreas with purified ab125027 at a working dilution of 1/400. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

All lanes : Anti-Histone H1.0 antibody [EPR6537] (ab125027) at 1/1000 dilution (unpurified)

Lane 1 : BxPC3 lysates
Lane 2 : Human fetal lung lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti rabbit at 1/2000 dilution

Predicted band size: 21 kDa

Unpurified ab125027, at a 1/100 dilution, staining Histone H1 in paraffin embedded Human kidney tissue by Immunohistochemistry.

Unpurified ab125027, at a 1/100 dilution, staining Histone H1 in A431 cells by Immunofluorescence.