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Knockout Tested Rabbit Recombinant Monoclonal HIF-1 alpha antibody. Suitable for ChIC/CUT&RUN-seq, IP, WB, ICC/IF, Flow Cyt (Intra), IHC-P and reacts with Human samples. Cited in 339 publications.


Images

Key facts

Isotype

IgG

Host species

Rabbit

Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form

Liquid

Clonality

Monoclonal

Immunogen

  • The exact immunogen used to generate this antibody is proprietary information.
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Reactivity data

Select an application
Product promiseTestedExpectedPredictedNot recommended
ChIC/CUT&RUN-seqIPWBICC/IFFlow Cyt (Intra)IHC-P
Human
Tested
Tested
Tested
Tested
Tested
Tested

Tested
Tested

Species

Human

Dilution info

5 µg

Notes

-

Tested
Tested

Species

Human

Dilution info

5 µg/mL

Notes

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Tested
Tested

Species

Human

Dilution info

1/100 - 1/1000

Notes

The antibody only works in hypoxic cell and tissue lysates.

For Mouse specific Hif-1-alpha rabbit monoclonal antibody, please see ab179483 (clone ID: EPR16897).

ab179483 has been confirmed for mouse samples in WB.

Compared with ab51608, ab308433 has higher sensitivity, we recommend ab308433 as an alternative for testing HIF-1 alpha in western blot.

Tested
Tested

Species

Human

Dilution info

1/500

Notes

-

Tested
Tested

Species

Human

Dilution info

1/10000

Notes

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Tested
Tested

Species

Human

Dilution info

1/100

Notes

For IHC antigen retrieval - See protocols .

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Target data

Function

Functions as a master transcriptional regulator of the adaptive response to hypoxia (PubMed:11292861, PubMed:11566883, PubMed:15465032, PubMed:16973622, PubMed:17610843, PubMed:18658046, PubMed:20624928, PubMed:22009797, PubMed:9887100, PubMed:30125331). Under hypoxic conditions, activates the transcription of over 40 genes, including erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, HILPDA, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia (PubMed:11292861, PubMed:11566883, PubMed:15465032, PubMed:16973622, PubMed:17610843, PubMed:20624928, PubMed:22009797, PubMed:9887100, PubMed:30125331). Plays an essential role in embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease (PubMed:22009797). Heterodimerizes with ARNT; heterodimer binds to core DNA sequence 5'-TACGTG-3' within the hypoxia response element (HRE) of target gene promoters (By similarity). Activation requires recruitment of transcriptional coactivators such as CREBBP and EP300 (PubMed:9887100, PubMed:16543236). Activity is enhanced by interaction with NCOA1 and/or NCOA2 (PubMed:10594042). Interaction with redox regulatory protein APEX1 seems to activate CTAD and potentiates activation by NCOA1 and CREBBP (PubMed:10202154, PubMed:10594042). Involved in the axonal distribution and transport of mitochondria in neurons during hypoxia (PubMed:19528298).(Microbial infection) Upon infection by human coronavirus SARS-CoV-2, is required for induction of glycolysis in monocytes and the consequent proinflammatory state (PubMed:32697943). In monocytes, induces expression of ACE2 and cytokines such as IL1B, TNF, IL6, and interferons (PubMed:32697943). Promotes human coronavirus SARS-CoV-2 replication and monocyte inflammatory response (PubMed:32697943).

Alternative names

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Knockout Tested Rabbit Recombinant Monoclonal HIF-1 alpha antibody. Suitable for ChIC/CUT&RUN-seq, IP, WB, ICC/IF, Flow Cyt (Intra), IHC-P and reacts with Human samples. Cited in 339 publications.

Alternative names

Key facts

Isotype

IgG

Form

Liquid

Clonality

Monoclonal

Immunogen
  • The exact immunogen used to generate this antibody is proprietary information.
Clone number

EP1215Y

Purification technique

Affinity purification Protein A

Specificity

This antibody recognizes HIF-1-alpha.For mouse specific Hif-1-alpha rabbit monoclonal antibody, please see ab179483 (clone ID: EPR16897). ab179483 has been confirmed for mouse samples in WB.

HIF-1 alpha expression is absent in most normal tissues (PMID: 12128120, 24835245, 11689469, 20217131).

Dissociation constant

2.24 x 10-10 M

Concentration
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Storage

Shipped at conditions

Blue Ice

Appropriate short-term storage duration

1-2 weeks

Appropriate short-term storage conditions

+4°C

Appropriate long-term storage conditions

-20°C

Aliquoting information

Upon delivery aliquot

Storage information

Avoid freeze / thaw cycle

Notes

For Mouse specific Hif-1-alpha rabbit monoclonal antibody, please see ab179483 (clone ID: EPR16897).

ab179483 has been confirmed for Mouse sample in WB.

We have mixed customer feedback towards the rat specificity so we are unable to confirm and guarantee its performance with rat samples. Please contact technical team for more information.

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

This product is a recombinant monoclonal antibody, which offers several advantages including:

  • - High batch-to-batch consistency and reproducibility
  • - Improved sensitivity and specificity
  • - Long-term security of supply
  • - Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

Biological function summary

HIF-1 alpha regulates gene expression in response to hypoxic conditions in cells. It forms a complex with HIF-1 beta to activate transcription of various genes involved in energy metabolism angiogenesis and erythropoiesis. HIF-1 alpha enables cells to adapt to reduced oxygen availability allowing for cellular survival and function under stress. It plays an important role in promoting the expression of genes like VEGF and EPO which are important for vascular and red blood cell development respectively.

Activity summary

HIF-1 alpha also known as hypoxia-inducible factor 1-alpha is a transcription factor critical in cellular response to low oxygen levels. Its molecular weight usually ranges from 93 to 120 kDa. You can find HIF-1 alpha expressed in tissues throughout the body but its expression significantly increases under hypoxic conditions. Researchers often use the HIF-1a ELISA to measure its expression levels. HIF-1 alpha forms a complex with other proteins to perform its functions effectively.

Pathways

HIF-1 alpha plays an integral role in the hypoxia signaling pathway and the glycolytic pathway. In the hypoxia signaling pathway HIF-1 alpha partners with VHL (Von Hippel-Lindau) protein that regulates its degradation under normal oxygen conditions. When oxygen levels drop HIF-1 alpha avoids degradation stabilizes and translocates into the nucleus to initiate transcription of hypoxia-responsive genes. The glycolytic pathway involvement highlights its function in adapting energy production under hypoxic conditions through collaboration with enzymes and transporters associated with glycolysis.

Associated diseases and disorders

HIF-1 alpha has been implicated in cancer and ischemic diseases. Its role in promoting angiogenesis and metabolic adaptation makes it a contributor to tumor growth and survival collaborating with oncogenes such as c-Myc. In ischemic diseases like stroke or myocardial infarction HIF-1 alpha's ability to induce protective responses can mitigate tissue damage through regulation of survival pathways. Understanding these interactions helps in the development of therapeutic strategies targeting HIF-1 alpha in disease contexts.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

23 product images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail
    Image from Chen L et al. HIF-1 alpha overexpression correlates with poor overall survival and disease-free survival in gastric cancer patients post-gastrectomy. PLoS One 9:e90678 (2014).

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    Immunohistochemical analysis of paraffin-embedded formalin-fixed human gastric cancer tissue stained for HIF-1 alpha using ab15608 at 1/600 dilution. Tissue sections were counterstained with Mayer's hematoxylin. Citrate buffer (pH 6.0) antigen retrieval using standard methodology

    C. HIF-1 alpha was located mainly in the nucleus of tumor cells (positive expression ×400).
    D. HIF-1 alpha original magnification ×100.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail
    Image from Zeindl-Eberhart E et al. Epithelial-mesenchymal transition induces endoplasmic-reticulum-stress response in human colorectal tumor cells. PLoS One 9:e87386 (2014).

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    Immunohistochemical analysis of Formalin-fixed paraffin-embedded human CRC tumour tissue using ab51608 for HIF-1 alpha staining. Endogenous peroxidase of sections was inhibited by 7.5% H2O2 at room temperature

    In central tumor areas of human CRCs β-catenin was typically localized at the cell membrane (A) whereas only a weak staining was observed for cytoplasmic GRP78 (B) and HIF-1 alpha staining was found to be negative (C). At the invasion front strong nuclear β-catenin was detectable indicating EMT (D, G). In corresponding regions strong cytoplasmic GRP78 expression was found (E, H). In some of the cases an intense nuclear HIF-1 alpha staining was observed (F, with hypoxia), but not in others (I, without hypoxia) (magnification 200×; scale bar: 100 μm).

  • Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail
    This image is courtesy of an anonymous customer review.

    Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

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

    All lanes: Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (AB51608) at 1/2000 dilution

    Lane 1: MCF-7 (normoxia) at 30000 Cells

    Lane 2: MCF-7 treated with 0.5% oxygen for 24 hours at 30000 Cells

    Secondary

    All lanes: Polyclonal Swine anti-rabbit IgG HRP at 1/1000 dilution

    Predicted band size: 92 kDa

  • Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    All lanes: Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (AB51608) at 1/100 dilution

    All lanes: Ramos Cells treated with Cocl2 at 10 µg

    Secondary

    All lanes: Goat Anti-Rabbit IgG, (H+L), HRP- conjugated at 1/1000 dilution

    Predicted band size: 92 kDa

  • Immunoprecipitation - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Immunoprecipitation - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    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

    All lanes: Immunoprecipitation - Anti-HIF-1 alpha antibody [EP1215Y] (AB51608)

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 92 kDa

    Observed band size: 110 kDa

    Exposure time: 12min

  • ChIC/CUT&RUN sequencing - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    ChIC/CUT&RUN sequencing - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 2.5 x 10^5 HeLa cells, and 5μg of ab51608 [EP1215Y]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads.
    Additional screenshots of mapped reads can be downloaded here.

  • Flow Cytometry (Intracellular) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Flow Cytometry (Intracellular) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    Overlay histogram showing HeLa untreated (Blue line) and HeLa treated (Red line - Deferoxamine, 1mM, 24 hours) cells stained with ab51608. 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, 1/11709 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr®488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C.

    Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

  • Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    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).

  • Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    ab51608 staining of HIF-1-alpha in untreated 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).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    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.

  • Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail
    Image courtesy of Teresa Otto (University of Duisburg-Essen, Germany)

    Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    Anti-HIF-1-alpha unpurified antibody (ab51608) reactivity with reduced Hep3B cell lysate after transient transfection of scrambled siRNA (lanes1-3 and 7-9) or HIF-1-alpha siRNA (lanes 4-6 and 10-12). Cells were incubated at with 21% O2 (lanes 1-6) or 1% O2 (lanes 7-12) for 4h before lysis. After SDS-PAGE, membranes were blocked in 5% milk for 1h at 25°C before incubation with unpurified ab51608 (1/1,000 dilution 5% milk) for 16h at 4°C. The blot was then incubated with an anti-Rabbit HRP-conjugated secondary antibody before developing with ECL.

    All lanes: Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (AB51608)

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 92 kDa

    Observed band size: ~120 kDa

    Exposure time: 6min

  • Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    Abcam recommends using 5% milk as the blocking agent, decreasing to 2% milk during primary and secondary incubation. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

    All lanes: Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (AB51608) at 1/2000 dilution

    Lane 1: Western blot - HeLa nuclear extract lysate (AB150036) at 40 µg

    Lane 2: Western blot - Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (AB180880) at 40 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 92 kDa

    Observed band size: 110 kDa

    Exposure time: 8min

  • Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    Abcam recommends using 5% milk as the blocking agent, decreasing to 2% milk during primary and secondary incubation. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

    All lanes: Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (AB51608) at 1/2000 dilution

    Lane 1: HeLa Whole Cell Lysate (untreated, negative control) at 40 µg

    Lane 2: HeLa DFO treated (0.5mM, 24h) Whole Cell Lysate at 40 µg

    Lane 3: HeLa Nuclear Cell Lysate (untreated, negative control) at 40 µg

    Lane 4: HeLa Nuclear DFO treated (0.5mM, 24h) Cell Lysate at 40 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 92 kDa

    Observed band size: 110 kDa

    Exposure time: 2min

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    Immunohistochemical analysis using unpurified ab51608 showing positive staining in Colonic adenocarcinoma tissue. Heat mediated antigen retrieval was performed via the microwave method before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    Immunohistochemical analysis using unpurified ab51608 showing positive staining in Squamous cell cervical carcinoma tissue. Heat mediated antigen retrieval was performed via the microwave method before commencing with IHC staining protocol.

  • OI-RD Scanning - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    OI-RD Scanning - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody.
    Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

  • ChIC/CUT&RUN sequencing - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    ChIC/CUT&RUN sequencing - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells treated with Cocl2 (500 μM 20h+4h) and MG-132 (10µM 4h) and 5 µg of ab51608 [EP1215Y]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

    Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.

    The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

  • Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    Western blot: Anti-HIF1A antibody [EP1215Y] (ab51608) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab51608 was shown to bind specifically to HIF1A. A band was observed at 105 kDa in wild-type Wild-type HCT 116 DMOG (0 mM, 4 h) cell lysates with no signal observed at this size in HIF1A knockout cell line. To generate this image, wild-type and HIF1A knockout Wild-type HCT 116 DMOG (0 mM, 4 h) cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

    All lanes: Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (AB51608) at 1/1000 dilution

    Lane 1: Wild-type HCT116 DMOG (0 mM, 4 h) cell lysate at 20 µg

    Lane 2: Wild-type HCT116 treated DMOG (1 mM, 4 h) cell lysate at 20 µg

    Lane 3: HIF1A knockout HCT116 DMOG (0 mM, 4 h) cell lysate at 20 µg

    Lane 4: HIF1A knockout HCT116 treated DMOG (1 mM, 4 h) cell lysate at 20 µg

    Secondary

    All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

    Performed under reducing conditions.

    Observed band size: 105 kDa

  • Flow Cytometry (Intracellular) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Flow Cytometry (Intracellular) - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    Flow cytometry overlay histogram showing left, HeLa treated with 1mM Deferoxamine for 24h and right, negative untreated HeLa stained with ab51608 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab51608) (1x 106 in 100μl at 0.2μg/ml (1/11000)) for 30min at 22°C.

    The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

    Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

    Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

    This antibody gave a positive signal in HeLa Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

  • Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    Western blot: Anti-HIF1A antibody [EP1215Y] (ab51608) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab51608 was shown to bind specifically to HIF1A. A band was observed at 100 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in HIF1A knockout cell line. To generate this image, wild-type and HIF1A knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

    All lanes: Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (AB51608) at 1/1000 dilution

    Lane 1: Wild-type HCT 116 DMOG (0 mM, 4 h) nuclear cell lysate at 20 µg

    Lane 2: Wild-type HCT 116 treated DMOG (1 mM, 4 h) nuclear cell lysate at 20 µg

    Lane 3: HIF1A knockout HCT 116 DMOG (0 mM, 4 h) nuclear cell lysate at 20 µg

    Lane 4: HIF1A knockout HCT 116 treated DMOG (1 mM, 4 h) nuclear cell lysate at 20 µg

    Lane 5: Wild-type HCT 116 DMOG (0 mM, 4 h) cell lysate at 20 µg

    Lane 6: Wild-type HCT 116 treated DMOG (1 mM, 4 h) cell lysate at 20 µg

    Lane 7: HIF1A knockout HCT 116 DMOG (0 mM, 4 h) cell lysate at 20 µg

    Lane 8: HIF1A knockout HCT 116 treated DMOG (1 mM, 4 h) cell lysate at 20 µg

    Secondary

    All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

    Performed under reducing conditions.

    Observed band size: 100 kDa

  • Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608), expandable thumbnail

    Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

    ab308433 1/1000
    ab181602 1/1000000

    Blocking/Diluting buffer and concentration: 5% NFDM/TBST
    Compared with ab51608, ab308433 has higher sensitivity, we recommend ab308433 as an alternative for testing HIF-1 alpha in western blot.

    Lanes 1 - 4: Western blot - Anti-HIF-1 alpha antibody [EP1215Y] (AB51608)

    Lanes 1 - 4: Western blot - Anti-HIF-1 alpha antibody [EP1215Y] - BSA and Azide free (AB210073)

    Lanes 1 and 3: Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

    Lane 2: HeLa treated with 0.5mM CoCl2 for 6h whole cell lysate at 20 µg

    Lane 4: HeLa treated with 0.5mM DFO for 24h whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051) at 1/20000 dilution

    Observed band size: 110 kDa

    Blocking/Diluting buffer and concentration: 5% NFDM/TBST
    Compared with ab51608, ab308433 has higher sensitivity, we recommend ab308433 as an alternative for testing HIF-1 alpha in western blot.

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