All lanes : Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (ab76020) at 1/100000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared from RIPA lysis method
Lane 2 : HeLa whole cell lysate prepared from 1% SDS HOT lysis method
Lane 3 : HeLa whole cell lysate prepared from RIPA lysis method
Lane 4 : HeLa whole cell lysate prepared from 1%SDS HOT lysis method
Lane 5 : Raw264.7 (Mouse abelson murine leukemia virus-induced tumor) whole cell lysate prepared from RIPA lysis method
Lanes 6 & 8 : Raw264.7 whole cell lysate prepared from 1%SDS HOT lysis method
Lane 7 : Raw264.7 whole cell lysate prepared from RIPA lysis method

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

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


Exposure time: 10 seconds

Blocking/Diluting buffer and concentration 5% NFDM/TBST

We suggest not to boil the sample after lysis.

T84 cells (human) 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.

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.

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.

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.

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.

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.

All lanes : Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (ab76020) at 1/100000 dilution

Lane 1 : CHO (Chinese hamster ovary cell line) cell lysate
Lane 2 : C6 (Rat glial tumor cell line) cell lysate
Lane 3 : Mouse brain

Lysates/proteins at 20 µg per lane.

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

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

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

We suggest not to boil the sample after lysis.

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

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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.

All lanes : Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (ab76020) at 1/100000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
Lane 3 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates
Lane 4 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution

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


Exposure time: 2 minutes

Blocking and diluting buffer: 5% NFDM/TBST.

We suggest not to boil the sample after lysis.

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.