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

• ICC/IF

Supplier Data

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 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). 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 ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• ICC/IF

Supplier Data

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), ab255394 cells labelling HIF-1 alpha with ab308433 at 1/100 (10.25 ug/ml) dilution, followed by 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). 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 ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt (Intra)

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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 (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, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ChIP-seq

Supplier Data

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 ab179483 [EPR16897]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• IP

Supplier Data

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) (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 : ab308433 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 (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 1/1000 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) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 110 kDa

false

Exposure time: 15s

• ChIP

Supplier Data

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 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 *Abcam Dual-X-ChIP protocol link

• ChIP-seq

Supplier Data

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 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-seq

Supplier Data

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 ab179483 [EPR16897]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• IP

Supplier Data

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) (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 : ab308433 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 (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 1/1000 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) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 110 kDa

false

Exposure time: 10s

• ICC/IF

Supplier Data

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 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). 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 ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt (Intra)

Supplier Data

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 (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, ab150081) at 1/5000 dilution was used as the secondary antibody.

• WB

Supplier Data

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 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 (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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 92 kDa

Observed band size: 110 kDa

false

Exposure time: 180s

• WB

Supplier Data

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 (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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 92 kDa

Observed band size: 110 kDa

true

Exposure time: 180s

• ChIC/CUT&RUN-seq

Lab

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 (350µM 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 ab172730 is also shown.

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

• ChIC/CUT&RUN-seq

Lab

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 (350µM 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 ab172730 is also shown.

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

• ChIC/CUT&RUN-seq

Lab

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 (350µM 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 ab172730 is also shown.

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