Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (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.

Image from Nieto-Torres JL et al. PLoS Pathog. 2014;10(5):e1004077. Fig 11.; doi: 10.1371/journal.ppat.1004077.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (AB76020)

Sodium Potassium ATPase immunofluorescence staining of MCF-7 cells using rabbit anti-Sodium Potassium ATPase antibody

Immunocytochemistry/Immunofluorescence analysis of MCF-7 (human breast carcinoma) cells labelling Sodium Potassium ATPase with purified ab76020 at 1/500 dilution. Cells were fixed with 100% methanol. ab150077 Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000 dilution) 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.

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (AB76020)

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.

Zhang B et al., PLoS One, 10, e0117976, 2015 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (AB76020)

Overlay histogram showing HeLa cells fixed in 80% methanoland 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.

• IHC-P

AbReview80742****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (AB76020)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of paraformaldehyde fixed human tonsil tissue permeabilized with 0.3% Triton X-100, staining with ab76020 at 5 µg/ml. Secondary used was ab150075 at 1/500 dilution. Blocking was done using Donkey Serum 10% + 3% BSA for 25 hours at 4°C. The sample was incubated with the primary antibody for 1 hour at 20°C. Antigen retrieval method was heat mediated Citrate pH 6 & TRIS pH 9. Validated on GE Cell DIVE.

This image is courtesy of an Abreview submitted by Dylan Windell

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (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 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5000 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (AB76020)

Immunohistochemical staining of paraffin embedded rat kidney with purified ab76020 at a dilution of 1/100. 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (AB76020)

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.

• ICC/IF

AbReview81949****

Immunocytochemistry/ Immunofluorescence - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (AB76020)

Immunocytochemistry analysis of formaldehyde-fixed rat hepatocytes permeabilized with 0.2% Triton X-100 in PBS staining with ab76020 at 1/50 dilution. Secondary antibody was Alexa Fluor™ 594 Donkey anti-Rb at 1/200 dilution.Cells were incubated with the primary antibody with 1% donkey serum in PBST for 2 hours at 22°C. Blocking was done with 1% donkey serum in PBST for 1 hour at 22°C.

This image is courtesy of an Abreview submitted by Armen Petrosyan

• IHC-P

AbReview79348****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (AB76020)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse prostate tissue permeabilized with 0.2% Triton X-100 in PBS. Stained with ab76020 at 1/50 dilution. Secondary antibody used was Biotinylated Goat Anti-Rabbit. Blocking was done with 1% ab64261 Rabbit specific HRP/DAB (ABC) Detection IHC Kit plus goat serum for 1 hour at 37°C. The sample was incubated with the primary antibody, diluted in Protein Block from ab64261 for 2 hours at 37°C. Antigen retrieval method was heat mediated with Tris-EDTA buffer (pH 9.0).

This image is courtesy of an Abreview submitted by Armen Petrosyan.

• WB

Unknown

Western blot - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (AB76020)

Blocking and diluting buffer : 5% NFDM/TBST.

We suggest not to boil the sample after lysis.

All lanes:

Western blot - 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 at 20 µg

Lane 2:

MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 3:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates at 20 µg

Lane 4:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates at 20 µg

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

false

Exposure time: 2min

• WB

Lab

Western blot - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (AB76020)

Blocking/Diluting buffer and concentration 5% NFDM/TBST

We suggest not to boil the sample after lysis.

All lanes:

Western blot - 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 at 15 µg

Lane 2:

HeLa whole cell lysate prepared from 1% SDS HOT lysis method at 15 µg

Lane 3:

HeLa whole cell lysate prepared from RIPA lysis method at 15 µg

Lane 4:

HeLa whole cell lysate prepared from 1%SDS HOT lysis method at 15 µg

Lane 5:

Raw264.7 (Mouse abelson murine leukemia virus-induced tumor) whole cell lysate prepared from RIPA lysis method at 15 µg

Lanes 6 and 8:

Raw264.7 whole cell lysate prepared from 1%SDS HOT lysis method at 15 µg

Lane 7:

Raw264.7 whole cell lysate prepared from RIPA lysis method at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 113 kDa

Observed band size: 100 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (AB76020)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

We suggest not to boil the sample after lysis.

All lanes:

Western blot - 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 at 20 µg

Lane 2:

C6 (Rat glial tumor cell line) cell lysate at 20 µg

Lane 3:

Mouse brain at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 113 kDa

Observed band size: 100 kDa

false