Anti-Sodium Potassium ATPase antibody [EP1845Y] - BSA and Azide free (ab167390) + MCF-7 (human breast carcinoma) whole cell lysate at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Predicted band size: 113 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?


Exposure time: 8 seconds

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM/TBST

T84 cells cultured on 8-well chamber slides, were washed once with ice-cold PBS, then fixed with 4% paraformaldehyde for 30 min at 4°C. After fixation, cells were permeabilized with 0.5% Triton X-100 for 5 min at room temperature and washed with PBS three times. Following blocking with 2% FCS in PBS for 1 hour at room temperature, primary antibody staining was performed at 4°C overnight at 1/200 dilution. Cells were then incubated with protein fractions B12 and C5 at 5x dilutions in fresh media for 1 hour at 37°C. Cells were then fixed, permeabilized and co-stained with fiber and sodium potassium ATPase. The nuclei were stained with DAPI using Vectachield mounting medium. Cells were visualized using Zeiss confocal microscopy LSM700.

Fiber molecules were found to be predominantly intracellularly following B12 treatment.

For full image see PubMed: 25723153.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76020).

Immunohistochemical staining of paraffin embedded human cervical carcinoma with purified ab76020 at a working dilution of 1 in 100. The secondary antibody used is a HRP conjugated goat anti-rabbit IgG (H+L), ab97051, at a dilution of 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76020).

Overlay histogram showing HeLa cells fixed in 80% methanol and stained with purified ab76020 at a dilution of 1 in 100 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 150. Rabbit monoclonal IgG was used as an isotype control (black line) and the blue line shows cells incubated without primary or secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76020).

Immunocytochemistry/Immunofluorescence analysis of MCF-7 (human breast carcinoma) cells labelling Sodium Potassium ATPase with purified ab76020 at 1/500. Cells were fixed with 100% methanol. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).

Secondary Only Control: PBS was used instead of the primary antibody as the negative control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76020).

ab76020 staining Sodium Potassium ATPase in lung epithelia (top) and bronchiolar epithelia (bottom) from Mouse lung tissue sections by Immunohistochemistry ((IHC) - paraffin-embedded sections). Sections were deparaffined at 60°C and rehydrated by successive incubations in 100% xylol, 100% ethanol and 96% ethanol. Samples were then permeabilized with 0.25% Triton X-100 in PBS for 15 minutes and blocked with 10% bovine serum albumin (BSA) and 0.25% Triton X-100 in PBS for 30 minutes. Samples were incubated with primary antibody (1/100 in 0.25% Triton X-100 and 10% BSA in PBS) for 1 hour 30 minutes at room temperature. An Alexa Fluor®488-conjugated Goat anti-mouse antibody was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76020).

Clone EP1845Y (ab167390) has been successfully conjugated by Abcam. This image was generated using Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (PE). Please refer to ab209299 for protocol details.

Overlay histogram showing HeLa cells stained with ab209299 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab209299, 1/2500 dilution) for 30 min at 22ºC.

Isotype control antibody (black line) was rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50mW Yellow-Green laser (561nm) and 586/15 bandpass filter.

This antibody gave a positive signal in HeLa cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

Clone EP1845Y (ab167390) has been successfully conjugated by Abcam. This image was generated using Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Marker (Alexa Fluor® 488). Please refer to ab197713 for protocol details.

ab197713 staining Sodium Potassium ATPase in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab197713 at a 1/50 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Clone EP1845Y (ab167390) has been successfully conjugated by Abcam. This image was generated using Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Marker (Alexa Fluor® 647). Please refer to ab198367 for protocol details.

ab198367 staining Sodium Potassium ATPase in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab198367 at a 1/100 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at a 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunohistochemical staining of paraffin embedded mouse liver with purified ab76020 at a working dilution of 1 in 100. The secondary antibody used is a HRP conjugated goat anti-rabbit IgG (H+L), ab97051, at a dilution of 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76020).

Immunohistochemical staining of paraffin embedded rat kidney with purified ab76020 at a working dilution of 1 in 100. The secondary antibody used is a HRP conjugated goat anti-rabbit IgG (H+L), ab97051, at a dilution of 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76020).

Immunohistochemical staining of Sodium Potassium ATPase in paraffin embedded human stomach carcinoma tissue with unpurified ab76020, at a 1/100 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76020).

Overlay histogram showing HeLa cells stained with unpurified ab76020 (red line). The cells were fixed with 80% methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab76020, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Please note that Abcam do not have any data for use of this antibody on non-fixed cells. We welcome any customer feedback.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76020).