Recombinant Anti-HIF-1 alpha antibody [EPR16897] (ab179483)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/µL, 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 ab179483 [EPR16897]. 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 downloaded here.

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

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.

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.

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.

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.

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).

Blocking and diluting buffer: 5% NFDM/TBST

The different result is due to the lysates preparation method. The lysate in the left image is prepared by RIPA lysis method. The lysate in the right image is prepared by 1%SDS Hot lysis method. This antibody works better in 1%SDS Hot Lysates in WB.

For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

Blocking and diluting buffer: 5% NFDM/TBST.

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Blocking/Dilution buffer: 5% NFDM/TBST.