Product nameHIF-1 alpha (H1alpha67, EP1215Y, ESEE122, EPR16897) Antibody Panel
See all HIF-1 alpha kits
Species reactivityReacts with: Human
HIF-1 alpha (H1alpha67, EP1215Y, ESEE122, EPR16897) Antibody Panel ab263460 contains multiple trial-sized versions of anti-human antibody clones against HIF-1 alpha, specifically selected for high performance in various applications. This panel contains 2 recombinant rabbit monoclonal antibodies and 2 mouse monoclonal antibodies against human HIF-1 alpha. They are provided as a sampler panel to allow you to easily evaluate each antibody.
For guidelines on how to use each antibody within the panel, please consult the individual datasheet for each antibody.
- Mouse monoclonal [H1alpha67] to HIF-1 alpha (20 µg) ab1
- Rabbit monoclonal [EP1215Y] to HIF-1 alpha (20 µL) ab51608
- Mouse monoclonal [ESEE122] to HIF-1 alpha (20 µg) ab8366
- Rabbit monoclonal [EPR16897] to HIF-1 alpha (20 µL) ab179483
HIF-1 alpha functions as a master transcriptional regulator of the adaptive response to hypoxia. Under such conditions, it can activate over 40 genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. (SwissProt: Q16665). Under normoxic conditions, HIF-1 alpha is largely undetectable and hypoxia will need to be induced in most cell lines and tissues before testing. Induction is typically not required for cancerous tissues where hypoxic regions are common within the tumor microenvironment (PubMed: 1850121, 11689469).
Explore our range of antibody sample panels designed to provide you with a variety of trial-size antibodies in a convenient and cost-effective format.
Directly conjugated versions of our antibodies are available and ready to use for multicolor flow cytometry or immunocytochemistry analysis. Please refer to the ‘Associated products’ section below.
Carrier-free formulations of our recombinant antibodies are also available for easy conjugation to labels of your choice and for multiplex applications. Please refer to the ‘Associated products’ section below.
Storage instructionsStore at -20°C. Please refer to protocols.
Components 1 kit ab51608 - Anti-HIF-1 alpha antibody 2 x 10µl ab179483 - Anti-HIF-1 alpha antibody [EPR16897] 2 x 10µl ab8366 - Anti-HIF-1 alpha antibody [ESEE122] 2 x 10µg ab1 - Anti-HIF-1 alpha antibody [H1alpha67] - ChIP Grade 2 x 10µg
FunctionFunctions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions activates the transcription of over 40 genes, including, erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. Plays an essential role in embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Activation requires recruitment of transcriptional coactivators such as CREBPB and EP300. Activity is enhanced by interaction with both, NCOA1 or NCOA2. Interaction with redox regulatory protein APEX seems to activate CTAD and potentiates activation by NCOA1 and CREBBP.
Tissue specificityExpressed in most tissues with highest levels in kidney and heart. Overexpressed in the majority of common human cancers and their metastases, due to the presence of intratumoral hypoxia and as a result of mutations in genes encoding oncoproteins and tumor suppressors.
Sequence similaritiesContains 1 basic helix-loop-helix (bHLH) domain.
Contains 1 PAC (PAS-associated C-terminal) domain.
Contains 2 PAS (PER-ARNT-SIM) domains.
DomainContains two independent C-terminal transactivation domains, NTAD and CTAD, which function synergistically. Their transcriptional activity is repressed by an intervening inhibitory domain (ID).
modificationsIn normoxia, is hydroxylated on Pro-402 and Pro-564 in the oxygen-dependent degradation domain (ODD) by EGLN1/PHD1 and EGLN2/PHD2. EGLN3/PHD3 has also been shown to hydroxylate Pro-564. The hydroxylated prolines promote interaction with VHL, initiating rapid ubiquitination and subsequent proteasomal degradation. Deubiquitinated by USP20. Under hypoxia, proline hydroxylation is impaired and ubiquitination is attenuated, resulting in stabilization.
In normoxia, is hydroxylated on Asn-803 by HIF1AN, thus abrogating interaction with CREBBP and EP300 and preventing transcriptional activation. This hydroxylation is inhibited by the Cu/Zn-chelator, Clioquinol.
S-nitrosylation of Cys-800 may be responsible for increased recruitment of p300 coactivator necessary for transcriptional activity of HIF-1 complex.
Requires phosphorylation for DNA-binding.
Sumoylated; by SUMO1 under hypoxia. Sumoylation is enhanced through interaction with RWDD3. Desumoylation by SENP1 leads to increased HIF1A stability and transriptional activity.
Ubiquitinated; in normoxia, following hydroxylation and interaction with VHL. Lys-532 appears to be the principal site of ubiquitination. Clioquinol, the Cu/Zn-chelator, inhibits ubiquitination through preventing hydroxylation at Asn-803.
The iron and 2-oxoglutarate dependent 3-hydroxylation of asparagine is (S) stereospecific within HIF CTAD domains.
Cellular localizationCytoplasm. Nucleus. Cytoplasmic in normoxia, nuclear translocation in response to hypoxia. Colocalizes with SUMO1 in the nucleus, under hypoxia.
- Information by UniProt
- ARNT interacting protein
- ARNT-interacting protein
- Basic helix loop helix PAS protein MOP1
- Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
- Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081)
- Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
- Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
- Goat Anti-Mouse IgG H&L (HRP) (ab205719)
- Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
- Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879)
- Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
- Alexa Fluor® 488 Anti-HIF-1 alpha antibody [EP1215Y] (ab190197)
- Alexa Fluor® 647 Anti-HIF-1 alpha antibody [EP1215Y] (ab190569)
- Alexa Fluor® 647 Anti-HIF-1 alpha antibody [H1alpha67] (ab203848)
- HRP Anti-HIF-1 alpha antibody [EP1215Y] (ab205833)
- Alexa Fluor® 488 Anti-HIF-1 alpha antibody [EPR16897] (ab208419)
- Alexa Fluor® 647 Anti-HIF-1 alpha antibody [EPR16897] (ab208420)
- Anti-HIF-1 alpha antibody [EP1215Y] - BSA and Azide free (ab210073)
- HRP Anti-HIF-1 alpha antibody [ESEE122] (ab215773)
- Anti-HIF-1 alpha antibody [EPR16897] - BSA and Azide free (ab221610)
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
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
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.
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab263460 has not yet been referenced specifically in any publications.