ll lanes : Anti-HIF-1 alpha antibody [EPR16897] (ab179483) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Wild type HAP1 treated with DMOG (0.5mM 18hr) whole cell lysate
Lane 3 : HIF1A knockout HAP1 whole cell lysate
Lane 4 : HIF1A knockout HAP1 treated with DMOG (0.5mM 18hr) whole cell lysate
Lane 5 : HeLa whole cell lysate
Lane 6 : HeLa treated with DMOG (0.5mM 18hr) whole cell lysate

Lysates/proteins at 40 µg per lane.

Predicted band size: 92 kDa
Observed band size: 105 kDa

Lanes 1 - 6: Merged signal (red and green). Green - ab179483 observed at 105 kDa. Red - loading control, ab24834, observed at 50 kDa.

ab179483 was shown to specifically react with HIF-1 alpha in wild-type HAP1 treated DMOG (0.5mM 18hr) cells as signal was lost in HAP1 knockout treated DMOG (0.5mM 18hr) knockout cells. Wild-type and HAP1 knockout samples were subjected to SDS-PAGE. ab179483 and ab24834 (Mouse anti-Histone H3 loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes: Anti-HIF-1 alpha antibody [EP1215Y] (ab51608) at 1/2000 dilution

Lane 1: MCF-7 (normoxia)
Lane 2: MCF-7 treated with 0.5% oxygen for 24 hours

Lysates/proteins at 30000 cells per lane.

Secondary for all lanes: Polyclonal Swine anti-rabbit IgG HRP at 1/1000 dilution

Predicted band size: 93 kDa


Blocking buffer: 5% milk for 16 hours at 4°C.

All lanes : Anti-HIF-1 alpha antibody [H1alpha67] - ChIP Grade (ab1) at 5 µg/ml

Lane 1 : HeLa nuclear extract lysate (ab150036) at 40 µg
Lane 2 : Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (ab180880) at 40 µg
Lane 3 : HeLa nuclear control at 40 µg
Lane 4 : HeLa nuclear DFO treated at 40 µg
Lane 5 : Recombinant Human HIF-1 alpha protein (ab154478) at 0.001 µg

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 92 kDa
Exposure time: 20 minutes


We recommend using 5% milk in TBST as the blocking agent, decreasing to 2% milk in TBST during primary and secondary antibody incubation.

Blots were developed with Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) secondary antibody

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling HIF-1-alpha with ab179483 at 1/50 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic and weak nuclear staining on HeLa cell line. The expression of HIF-1-alpha was increased upon CoCl2 treatment (0.6nM for 6 hours); and the protein also translocated from the cytoplasm into the nucleus. [Biochimica et Biophysica Acta 1745 (2005) 187 – 195.] The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
1. ab179483 at 1/50 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor^® 488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of hypoxia-induced Human placenta labeling HIF-1-alpha with ab8366.

ab8366 stained Hela cells. The cells were 4% formaldehyde fixed for 10 minutes, permeabilized in 0.1% PBS-Triton X-100 for 5 min and then blocked in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour at room temperature to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab8366 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed  used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

ab51608 staining HIF-1-alpha in Human ovarian carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed and paraffin-embedded, antigen retrieval was by heat mediation in Tris/EDTA buffer pH9. Samples were incubated with primary antibody (1/100). An undiluted HRP-conjugated anti-rabbit IgG was used as the secondary antibody. Tissue counterstained with Hematoxylin.

HeLa cells were untreated or treated with 1mM Deferoxamine (DFO) for 24h and fixed with paraformaldehyde for imaging by fluorescent microscopy. Cells were blocked and stained with 1X blocking buffer (ab126587). Unpurified ab51608 was used at 1:500. DAPI was used to label the nucleus. HIF1 alpha staining is absent in untreated cells and induced by DFO treatment. HIF1 alpha localizes to the nucleus.

ab1 staining HIF-1-alpha in MCF7 (Human breast adenocarcinoma cell line) cells treated with metformin hydrochloride ab12084, by ICC/IF. Decrease in HIF-1-alpha expression correlates with increased concentration of metformin hydrochloride, as described in literature.
The cells were incubated at 37°C for 24 hours in media containing different concentrations of ab120847 (metformin hydrochloride) in water, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2 hours at room temperature. Staining of the treated cells with ab1 (10 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. Goat Anti-Mouse IgG H&L (DyLight^® 488) preadsorbed (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.