ab7671 staining alpha 1 Sodium Potassium ATPase in Hek293 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween 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 ab7671 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.

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.

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

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.