Anti-HIF-1 alpha antibody [EP1215Y]

24 product images

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  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] (Anti-alpha Tubulin antibody [DM1A] - Loading Control 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

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  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.

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  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).

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  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

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  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

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

  HIF-1-alpha was immunoprecipitated using 0.5mg HeLa Nuclear DFO treated whole cell extract (Hela-DFO treated (0.5mM, 24h) Nuclear Lysate 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) (Mouse monoclonal [SB62a] Anti-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

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

  CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse 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.

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  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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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.

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  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).

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

  Unpurified ab51608 staining HIF-1-alpha in HepG2 cells treated with baicalein (Baicalein, Lipoxygenase inhibitor. 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 Baicalein, Lipoxygenase inhibitor. 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 (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/250 dilution was used as the secondary antibody.

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  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).

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  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.

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  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 (HeLa nuclear extract lysate ab150036) at 40 µg

  Lane 2: Western blot - Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (Hela-DFO treated (0.5mM, 24h) Nuclear Lysate ab180880) at 40 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (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

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  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

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  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) (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

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  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.
  
  

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  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.

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  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.

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  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.

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  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.

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

  Anti-HIF-1 alpha antibody [EPR16897-145] ab308433 1/1000
  Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 1/1000000
  
  Blocking/Diluting buffer and concentration: 5% NFDM/TBST
  Compared with ab51608, Anti-HIF-1 alpha antibody [EPR16897-145] ab308433 has higher sensitivity, we recommend Anti-HIF-1 alpha antibody [EPR16897-145] 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 (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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 110 kDa

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  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] (Anti-alpha Tubulin antibody [DM1A] - Loading Control 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

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  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 Rabbit IgG, monoclonal [EPR25A] - Isotype 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.

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

  HIF-1 alpha Immunocytochemistry/ Immunofluorescence staining of HeLa cells using rabbit Anti-HIF-1 alpha antibody

  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 (10X 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.