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

ab7671 staining alpha 1 Sodium Potassium ATPase in human formaldehyde tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 5% BSA for 12 hours at 4°C; antigen retrieval was by heat mediation in a buffer pH 9. Samples were incubated with primary antibody (5µg/ml in 5% BSA) for 16 hours at 4°C. An Alexa Fluor^® 488-conjugated donkey anti-mouse IgG polyclonal (1/1000) was used as the secondary antibody.

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Lanes 1-3 : Anti-alpha 1 Sodium Potassium ATPase antibody [464.6] (ab7671) at 1 µg/ml
Lanes 4-6 : Anti-alpha 1 Sodium Potassium ATPase antibody [464.6] (ab7671) at 2 µg/ml
Lanes 7-9 : Anti-alpha 1 Sodium Potassium ATPase antibody [464.6] (ab7671) at 5 µg/ml

Lanes 1 & 4 & 7 : Human brain tissue lysate - total protein (ab29466)
Lanes 2 & 5 & 8 : Brain (Mouse) Tissue Lysate (ab27253)
Lanes 3 & 6 & 9 : Brain (Rat) Tissue Lysate (ab7942)

Lysates/proteins at 20 µg per lane.

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 112 kDa


Exposure time: 3 minutes

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.

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 Donkey Anti-Mouse IgG H&L (Phycoerythrin) preadsorbed (ab7003) secondary antibody, 5 µg/ml, was used.

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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 : Human kidney tissue lysate - total protein (ab30203)
Lane 3 : Heart (Rabbit) Whole Cell Lysate - normal tissue (ab29072)
Lane 4 : Human brain tissue lysate - total protein (ab29466)
Lane 5 : Brain (Human) Membrane Lysate - adult normal tissue

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : 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

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.

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

Predicted band size: 112 kDa



Western blot analysis detecting Na, K-ATP-ase (alpha) in porcine proximal tubule protein, using Ab7671. Band is at 112 kDa.

ICC/IF image of ab7671 stained Hek293 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 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.

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

All lanes : Parotid acinar cell lysate

Lysates/proteins at 50 µg per lane.

Secondary
All lanes : 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

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Immunocytochemistry/ Immunofluorescence analysis of B16 mice tumor cells labeling alpha 1 Sodium Potassium ATPase with ab7671 at 1/200 dilution. Cells were fixed in formaldehyde and permeabilized with 0.25% Triton-X100 for 10 minutes. Cells were blocked with 1% BSA for 1 hour at 20°C, followed by staining with ab7671 at 1/200 for 18 hours at 4°C in 1% BSA in PBST buffer. Goat Anti-Mouse IgG H&L (Alexa Fluor® 555) preadsorbed (ab150118) secondary antibody was used at 1/1000 dilution. DAPI was used to counterstain.

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