Anti-alpha 1 Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker (ab7671)

  Western blot - alpha 1 Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker (ab7671)

All lanes : alpha 1 Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker at 10 µg/ml

Lane 1 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 2 : Kidney (Human) Tissue Lysate - adult normal tissue (ab30203)
Lane 3 : Heart (Rabbit) Whole Cell Lysate - normal tissue (ab29072)
Lane 4 : Brain (Human) Tissue Lysate - adult normal tissue (ab29466)
Lane 5 : Brain (Human) Membrane Lysate - adult normal tissue

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 112 kDa
Observed band size : 100 kDa (why is the actual band size different from the predicted?)


Exposure time : 3 minutes

  Western blot - Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker (ab7671)

Anti-alpha 1 Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker (ab7671) at 1/5000 dilution + Porcine proximal tubule lysate

Predicted band size : 112 kDa

  Immunocytochemistry/ Immunofluorescence - Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker (ab7671)

ab7671 staining (dog) MDCK II cells by ICC/IF.  Cells were acetone fixed and blocked with 4% serum for 10 minutes at 25°C.  The primary antibody was diluted 1/100 and incubated with the sample for 16 hours at 4°C.  Ab6785 (a FITC conjugated goat polyclonal to mouse IgG - H&L) was diluted 1/100 and employed as the secondary antibody.

This image is courtesy of an anonymous Abreview

See Abreview

  Flow Cytometry - Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker (ab7671)

ab7671 staining Sodium Potassium ATPase - Plasma Membrane Marker in Human Fibrosarcoma HT1080 cells by Flow Cytometry. Cells were fixed with paraformaldehyde. The  sample was incubated with the primary antibody 10 µg/ml in PBS  for 1 hour. An Abcam PE-conjugated donkey polyclonal to mouse IgG ( ab7003), 5 µg/ml, was used as secondary antibody.

This image is courtesy of an anonymous Abreview

See Abreview

  Immunocytochemistry/ Immunofluorescence - Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker (ab7671)

ab7671 staining Sodium Potassium ATPase in 293 human embryonic kidneys cells. Cells were plated on an uncoated cover slip. One day after plating cells were washed twice with PBS and fixed with 4% paraformaldehyde with 4% sucrose at room temperature for 15 minutes. After washing once with PBS, 300µl wheat germ agglutinin (5µg/ml in PBS) conjugated to TexasRed were added and incubated at 4°C in darkness for 15 minutes. Cells were then washed twice with PBS and permeabilized with 0.5% saponin in PBS for 45 minutes at 4°C. Blocking was carried out with 10% fetal calf serum in PBS-saponin during first and second antibody incubation. An Alexa Fluor 488^® conjugated goat polyclonal to mouse IgG was used as the secondary antibody at a 1/500 dilution. After additional washing cover slips were mounted on glass slides with ProLong^® Gold antifade reagent with DAPI.

This image is a courtesy of Anonymous Abreview

See Abreview

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - alpha 1 Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker (ab7671)

IHC image of Ab7671 staining in Human Kidney Carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM 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 Ab7671, 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.

  Immunocytochemistry/ Immunofluorescence - alpha 1 Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker (ab7671)

ICC/IF image of ab7671 stained Hek293 cells. The cells were 4% Hek293 fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7671, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa, HepG2 and MCF7 cells at 5µg/ml.

  Immunocytochemistry/ Immunofluorescence - alpha 1 Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker (ab7671)

ICC/IF image of ab7671 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7671, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-mouse IgG - H&L, pre-adsorbed (ab96879) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  Western blot - Anti-alpha 1 Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker (ab7671)

All lanes : Anti-alpha 1 Sodium Potassium ATPase antibody [464.6] - Plasma Membrane Marker (ab7671) at 1/1000 dilution

Lane 1 : Parotid acinar cell lysate
Lane 2 : Parotid acinar cell lysate

Lysates/proteins at 50 µg per lane.

Secondary
HRP-conjugated Donkey anti-Mouse polyclonal at 1/10000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 112 kDa
Observed band size : 112 kDa


Exposure time : 5 minutes

See Abreview