Anti-HIF-1-alpha antibody [EP1215Y]

• Supplier: Abcam
• Catalog: ab51608
• Prices: $403.00
• Sizes: 100 µl
• Host: Rabbit
• Clonality: Monoclonal
• Isotype: IgG
• Clone: EP1215Y
• Applications: ICC/IF ICC/IF FC IP WB IHC-P
• Species Reactivities: Mouse, Rat, Human
• Antigen: Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human HIF-1-alpha aa 600-700 (C terminal)
• Blocking Peptide: HIF-1-alpha peptide
• Description: Rabbit Monoclonal
• Gene: HIF1A
• Conjugate: Unconjugated

Product images

ab51608 staining HIF-1-alpha in HeLa cell line treated with Cocl2 by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody (1/500). An Alexa Fluor®488-conjugated Goat anti-rabbit IgG(1/200) was used as the secondary antibody. Nuclei were counterstained with DAPI(right hand image).

ab51608 staining HIF-1-alpha in HeLa cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody (1/500). An Alexa Fluor®488-conjugated Goat anti-rabbit IgG(1/200) was used as the secondary antibody. Nuclei were counterstained with DAPI(right hand Image).

ab51608 staining HIF-1-alpha in mouse kidney 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.

ab51608 staining HIF-1-alpha in rat muscle 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.

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.

Anti-HIF-1-alpha antibody [EP1215Y] (ab51608) at 1/100 dilution + Ramos Cells treated with Cocl2 at 10 µgSecondaryGoat Anti-Rabbit IgG, (H+L), HRP- conjugated at 1/1000 dilution

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.

Unpurified ab51608 staining HIF-1-alpha in HepG2 cells treated with baicalein (ab120723), by ICC/IF. Increase in HIF-1-alpha expression correlates with increased concentration of baicalein as described in literature.The cells were incubated at 37°C for 6h in media containing different concentrations of ab120723 (baicalein) in DMSO, 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 2h at room temperature. Staining of the treated cells with ab51608 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody.

Immunohistochemical analysis using unpurified ab51608 showing positive staining in Breast carcinoma tissue.

Immunohistochemical analysis using unpurified ab51608 showing positive staining in Colonic adenocarcinoma tissue.

Immunohistochemical analysis using unpurified ab51608 showing positive staining in Squamous cell cervical carcinoma tissue.

All lanes : Anti-HIF-1-alpha antibody [EP1215Y] (ab51608) at 1/2000 dilution (Unpurified)Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab150036)Lane 2 : Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (ab180880)Lysates/proteins at 40 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.

HIF-1-alpha was immunoprecipitated using 0.5mg HeLa Nuclear DFO treated whole cell extract (ab180880), 5µg of Rabbit polyclonal to HIF1 alpha and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).The antibody was incubated under agitation with Protein G beads for 10min, HeLa DFO treated whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with unpurified ab51608.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 110kDa; HIF1 alpha

developed using the ECL techniquePerformed under reducing conditions.

All lanes : Anti-HIF-1-alpha antibody [EP1215Y] (ab51608) at 1/2000 dilution (unpurified)Lane 1 : HeLa Whole Cell Lysate (untreated, negative control)Lane 2 : HeLa DFO treated (0.5mM, 24h) Whole Cell LysateLane 3 : HeLa Nuclear Cell Lysate (untreated, negative control)Lane 4 : HeLa Nuclear DFO treated (0.5mM, 24h) Cell LysateLysates/proteins at 40 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilutionPerformed under reducing conditions.

Overlay histogram showing HeLa cells stained with unpurified ab51608 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab51608, 1:50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

Equilibrium disassociation constant (KD)Learn more about KD Click here to learn more about KD

Product References

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HIF-1 alpha overexpression correlates with poor overall survival and disease-free - HIF-1 alpha overexpression correlates with poor overall survival and disease-free

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BAG3 and HIF-1 alpha coexpression detected by immunohistochemistry correlated - BAG3 and HIF-1 alpha coexpression detected by immunohistochemistry correlated

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