Anti-HIF-1 alpha antibody [EPR16897]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [EPR16897] (AB179483)

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 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in HeLa cells treated with DFO (1 mM, 24 h). The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 (anti-alpha Tubulin mouse mAb) (Alexa Fluor^® 594) at 1/200 dilution (red).

• IP

Lab

Immunoprecipitation - Anti-HIF-1 alpha antibody [EPR16897] (AB179483)

ab179483 at 1/30 dilution immunoprecipitating HIF-1 alpha in HeLa (human cervix adenocarcinoma epithelial cell) treated with 500 µM CoCl2 for 24 hours whole cell lysate. Western blot was performed from the immunoprecipitate using ab179483 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution. Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) treated with 500 µM CoCl2 for 24 hours whole cell lysate 10 µg Lane 2 : ab179483 IP in HeLa (human cervix adenocarcinoma epithelial cell) treated with 500 µM CoCl2 for 24 hours whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab179483 in HeLa treated with 500 µM CoCl2 for 24 hours whole cell lysate Blocking/Dilution buffer : 5% NFDM/TBST. Exposure time : 8 seconds.

Lane 2:

Immunoprecipitation - Anti-HIF-1 alpha antibody [EPR16897] (ab179483) at 1/30 dilution

Lane 2:

Immunoprecipitation - Anti-HIF-1 alpha antibody [EPR16897] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/hif-1-alpha-antibody-epr16897-bsa-and-azide-free-ab221610'>ab221610</a>)

All lanes:

HeLa (human cervix adenocarcinoma epithelial cell) treated with 500 µM CoCl2 for 24 hours 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: 8s

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-HIF-1 alpha antibody [EPR16897] (AB179483)

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] (AB179483)

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] (AB179483)

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.

• WB

Supplier Data

Western blot - Anti-HIF-1 alpha antibody [EPR16897] (AB179483)

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

The expression profile observed is consistent with what has been described in the literature (PMID : 17906695).

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

In Western Blot, Anti HIF-1 alpha antibody [EPR16897-101] (ab179483) is applied at 1/1000 dilution to prove the DFO induction is successful.

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

All lanes:

Western blot - Anti-CITED2 antibody [EPR27399-62] (<a href='/en-us/products/primary-antibodies/cited2-antibody-epr27399-62-ab314757'>ab314757</a>) at 1/1000 dilution

Lane 1:

Untreated HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HT-1080 treated with 0.5 mM DFO for 24 hours whole cell lysate at 20 µg

Lane 3:

HT-1080 treated with 0.5 mM DFO for 24 hours, then added 10uM MG-132 for 4 hours, whole cell lysate at 20 µg

Lane 4:

HT-1080 treated with 10uM MG-132 for 4 hours, 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

Observed band size: 30 kDa,36 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-HIF-1 alpha antibody [EPR16897] (AB179483)

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

Lysates were freshly made and used immediately to minimise protein degradation.

Lanes 1 - 4:

Western blot - Anti-HIF-1 alpha antibody [EPR16897] (ab179483) at 1/1000 dilution

Lanes 1 - 4:

Western blot - Anti-HIF-1 alpha antibody [EPR16897] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/hif-1-alpha-antibody-epr16897-bsa-and-azide-free-ab221610'>ab221610</a>)

Lane 1:

Untreated HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HT-1080 treated with 0.5 mM DFO for 24 hours whole cell lysate at 20 µg

Lane 3:

HT-1080 treated with 0.5 mM DFO for 24 hours, then added 10uM MG-132 for 4 hours, whole cell lysate at 20 µg

Lane 4:

HT-1080 treated with 10uM MG-132 for 4 hours, whole cell lysate at 20 µg

Observed band size: 50-110 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-HIF-1 alpha antibody [EPR16897] (AB179483)

Lanes 1 - 6 : Merged signal (red and green). Green - ab179483 observed at 105 kDa. Red - loading control, ab24834, observed at 50 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 (0.5 mM 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:

Western blot - Anti-HIF-1 alpha antibody [EPR16897] (ab179483) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 40 µg

Lane 2:

Wild type HAP1 treated with DMOG (0.5mM 18hr) whole cell lysate at 40 µg

Lane 3:

HIF1A knockout HAP1 whole cell lysate at 40 µg

Lane 4:

HIF1A knockout HAP1 treated with DMOG (0.5mM 18hr) whole cell lysate at 40 µg

Lane 5:

HeLa whole cell lysate at 40 µg

Lane 6:

HeLa treated with DMOG (0.5mM 18hr) whole cell lysate at 40 µg

Predicted band size: 92 kDa

Observed band size: 105 kDa

false

• WB

Lab

Western blot - Anti-HIF-1 alpha antibody [EPR16897] (AB179483)

HIF-1-alpha is constantly synthesized but rapidly degraded under normoxic conditions, whereas reduced oxygen concentration results in stabilization of HIF-1-alpha. (PMID : 15104534)

Exposure time : Lanes 1-4 : 10 seconds, Lanes 5-6 : 20 seconds

All lanes:

Western blot - Anti-HIF-1 alpha antibody [EPR16897] (ab179483) at 1/1000 dilution

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

Lane 5:

Untreated NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 6:

NIH/3T3 (Mouse embryonic fibroblast) treated with 0.1mM CoCl2 for 48h 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 1/20000 dilution

Observed band size: 50-110 kDa

false

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-HIF-1 alpha antibody [EPR16897] (AB179483)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HaCaT (Human keratinocyte cell line) cells (treated with 7ng/ml TGF-β for 1h) and 5 µg of ab40855 [EP784Y]. 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 Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Lab

Western blot - Anti-HIF-1 alpha antibody [EPR16897] (AB179483)

Blocking and diluting buffer : 5% NFDM/TBST.

The expression of HIF-1 alpha is induced by CoCl2 and maintained by MG-132 (PMID : 15836611).

All lanes:

Western blot - Anti-HIF-1 alpha antibody [EPR16897] (ab179483) at 0.163 µg/mL

Lane 1:

Untreated C6 (rat glial tumor glial cell), whole cell lysate at 10 µg

Lane 2:

C6 treated with 400 &mu;M CoCl2 and 20 &mu;M MG-132 (<a href='/en-us/products/biochemicals/mg-132-proteasome-inhibitor-ab141003'>ab141003</a>) for 4 hours at 10 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 1/20000 dilution

Predicted band size: 92 kDa

Observed band size: 110 kDa

false

Exposure time: 26s