Anti-HIF-1 alpha antibody [EPR16897-145]

16 product images

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  ChIP - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  Chromatin was prepared from HeLa (human cervical adenocarcinoma epithelial cell) treated with DFO(0.5mM 24h) and HeLa non-treated according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
  The ChIP was performed with 25 µg of chromatin, 5 µg of ab308433 (red), or 5 µg of rabbit normal IgG Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
  Primers are from paper PMID:28193910, 21447827
  *http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

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  Western blot - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  

  The expression of HIF-1 alpha is upregulated in response to DFO treatment (PMID: 25075254).

  In Western blot, anti-GAPDH antibody (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/200000 dilution.

  This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

  Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

  All lanes: Western blot - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433) at 1/1000 dilution

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

  Lane 2: HeLa treated with 0.5 mM DFO for 24 hour 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 1/100000 dilution

  Developed using the ECL technique.

  Predicted band size: 92 kDa

  Observed band size: 110 kDa

  Exposure time: 180s

• 

  ChIC/CUT&RUN sequencing - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with CoCl2 (350?M 20h+4h) add MG-132(10µM 4h) and 5 µg of ab308433 [EPR16897-145]. 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.

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

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  ChIC/CUT&RUN sequencing - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with CoCl2 (350?M 20h+4h) add MG-132(10µM 4h) and 5 µg of ab308433 [EPR16897-145]. 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.

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

• 

  ChIC/CUT&RUN sequencing - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with CoCl2 (350?M 20h+4h) add MG-132(10µM 4h) and 5 µg of ab308433 [EPR16897-145]. 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.

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

• 

  Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized bEnd.3 (mouse brain endothelial cell) cells labelling HIF-1 alpha with ab308433 at 1/100 (5.125 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal images showing nuclear staining in bEnd.3 cells treated with DFO (2 mM) for 24 hours and showing no staining in untreated bEnd.3 cells.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

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  Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HIF1A KO HCT 116 (HIF1A knockout human colorectal carcinoma epithelial cell), Human HIF1A (HIF-1 alpha) knockout HCT116 cell line ab255394 cells labelling HIF-1 alpha with ab308433 at 1/100 (10.25 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal images showing nuclear staining in wildtype HCT 116 cells treated with DFO (0.5mM) for 24 hours and showing no staining in HIF1A knockout HCT 116 cells treated with the same condition.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling HIF-1 alpha with ab308433 at 1/100 (5.125 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal images showing nuclear staining in HeLa cells treated with DFO (0.5 mM) for 24 hours and showing no staining in untreated HeLa cells.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

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  ChIP-sequencing - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  Chromatin was prepared from HeLa cells treated with CoCl2 (350μM 20h+4h) add MG-132(10µM 4h). Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 µg of ab308433 [EPR16897-145] or Anti-HIF-1 alpha antibody [EPR16897] ab179483 [EPR16897]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

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  ChIP-sequencing - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  Chromatin was prepared from HeLa cells treated with CoCl2 (350μM 20h+4h) add MG-132(10µM 4h). Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 µg of ab308433 [EPR16897-145] or Anti-HIF-1 alpha antibody [EPR16897] ab179483 [EPR16897]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• 

  ChIP-sequencing - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  Chromatin was prepared from HeLa cells treated with CoCl2 (350μM 20h+4h) add MG-132(10µM 4h). Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 µg of ab308433 [EPR16897-145] or Anti-HIF-1 alpha antibody [EPR16897] ab179483 [EPR16897]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• 

  Immunoprecipitation - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  HIF-1 alpha was immunoprecipitated from 0.35 mg HCT116 (human colorectal carcinoma epithelial cell) treated with 0.5 mM DFO for 24 hour whole cell lysate with ab308433 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab308433 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
  
  Lane 1: HCT116 (human colorectal carcinoma epithelial cell) treated with 0.5 mM DFO for 24 hour whole cell lysate
  
  Lane 2: abAB308433 IP in HCT116 (human colorectal carcinoma epithelial cell) treated with 0.5 mM DFO for 24 hour whole cell lysate
  
  Lane 3:Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab308433 in HCT116 treated with 0.5 mM DFO for 24 hour whole cell lysate
  
  Blocking and dilution buffer and concentration: 5% NFDM/TBST.
  
  Exposure time: 15 seconds

  All lanes: Immunoprecipitation - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433) at 1/30 dilution

  All lanes: HCT116 (human colorectal carcinoma epithelial cell) treated with 0.5 mM DFO for 24 hour whole cell lysate at 10 µg

  Secondary

  All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution

  Observed band size: 110 kDa

  Exposure time: 15s

• 

  Immunoprecipitation - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  HIF-1 alpha was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) treated with 0.5 mM DFO for 24 hour whole cell lysate with ab308433 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab308433 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
  
  Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) treated with 0.5 mM DFO for 24 hour whole cell lysate
  
  Lane 2: abAB308433 IP in HeLa (human cervix adenocarcinoma epithelial cell) treated with 0.5 mM DFO for 24 hour whole cell lysate
  
  Lane 3:Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab308433 in HeLa treated with 0.5 mM DFO for 24 hour whole cell lysate
  
  Blocking and dilution buffer and concentration: 5% NFDM/TBST.
  
  Exposure time: 10 seconds

  All lanes: Immunoprecipitation - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433) at 1/30 dilution

  All lanes: HeLa (human cervix adenocarcinoma epithelial cell) treated with 0.5 mM DFO for 24 hour whole cell lysate at 10 µg

  Secondary

  All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution

  Observed band size: 110 kDa

  Exposure time: 10s

• 

  Flow Cytometry (Intracellular) - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell) treated with 0.5mM DFO for 24h (Red) / untreated HeLa (Green) cells labelling HIF-1 alpha with ab308433 at 1/50 dilution (1 ug)/Red and Green (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/5000 dilution was used as the secondary antibody.

• 

  Flow Cytometry (Intracellular) - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized bEND.3 (mouse brain endothelial cell) treated with 2mM DFO for 24h (Red) / untreated bEND.3 (Green) cells labelling HIF-1 alpha with ab308433 at 1/50 dilution (1 ug)/Red and Green (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/5000 dilution was used as the secondary antibody.

• 

  Western blot - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.#Lysates/proteins at 20 µg per lane.
  
  Performed under reducing conditions.
  
  In Western blot, ab308433 was shown to bind specifically to HIF-1 alpha. A band was observed at 110 kDa in wild-type HCT116 cell lysates with whereas no signal observed at this size in HIF-1 alpha knockout cell line Human HIF1A (HIF-1 alpha) knockout HCT116 cell line ab255394 (knockout cell lysate).
  
  The expression of HIF-1 alpha is upregulated in response to DFO treatment (PMID: 25075254).
  
  In Western blot, anti-GAPDH antibody (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/200000 dilution.
  
  Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.
  
  Exposure time: 180 seconds.

  All lanes: Western blot - Anti-HIF-1 alpha antibody [EPR16897-145] (ab308433) at 1/1000 dilution

  Lane 1: Untreated HCT116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: HCT116 treated with 0.5 mM DFO for 24 hour whole cell lysate at 20 µg

  Lane 3: Untreated HIF-1 alpha knockout HCT116 whole cell lysate at 20 µg

  Lane 4: HIF-1 alpha knockout HCT116 treated with 0.5 mM DFO for 24 hour whole cell lysate at 20 µg

  Lane 5: Untreated bEnd.3 (mouse brain endothelioma) whole cell lysate at 20 µg

  Lane 6: bEnd.3 treated with 0.5 mM DFO for 24 hour 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 1/100000 dilution

  Predicted band size: 92 kDa

  Observed band size: 110 kDa

  Exposure time: 180s