All lanes : Anti-HIF-2-alpha antibody (ab109616) at 1 µg/ml

Lane 1 : Wild-type A549 untreated cell lysate
Lane 2 : Wild-type A549 + DFO (1mM, 24 hours) cell lysate
Lane 3 : EPAS knockout A549 untreated cell lysate
Lane 4 : EPAS knockout A549 + DFO (1mM, 24 hours) cell lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Predicted band size: 96 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab109616 observed at 100 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab109616 was shown to react with HIF-2-alpha in A549 wild-type cells in western blot with loss of signal observed in EPAS1 knockout sample. A549 wild-type and EPAS1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab109616 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

IHC image of HIF-2alpha staining in a section of formalin-fixed paraffin-embedded normal human colon*, performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 for 20mins. The section was then incubated with ab109616 at 1μg/ml, for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

ab109616 staining Hif-2-alpha in HeLa cells. The cells were incubated with 1mM Deferoxamine for 24 hours (Treated) or solvent-only for control purposes (Solvent only). Cells were fixed with 4% formaldehyde (10min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab109616 at 10µg/ml and ab195889 at 1µg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI. 

All lanes : Anti-HIF-2-alpha antibody (ab109616) at 1 µg/ml (Milk blocking - 3%)

Lane 1 : Human placenta tissue lysate - total protein (ab29745)
Lane 2 : A549 (Human lung adenocarcinoma epithelial cell line) Whole Cell Lysate
Lane 3 : Human heart tissue lysate - total protein (ab29431)
Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 96 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?
Additional bands at: 100 kDa, 38 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 16 minutes


The predicted molecular weight of HIF2 alpha is 96 kDa (SwissProt), however we expect to observe a banding pattern around 120 kDa. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.