Anti-HIF-1 alpha antibody [54/HIF-1a]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [54/HIF-1a] (AB279654)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling HIF-1 alpha with ab279654 at 1/20 dilution, followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green).

Confocal image showing nuclear staining in HeLa cells treated with 1 mM desferrioxamine for 24 hrs.

ab179513 anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) at 1/1000 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only controls : Secondary antibodies are ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution and ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) at 1/1000 dilution.

• WB

Lab

Western blot - Anti-HIF-1 alpha antibody [54/HIF-1a] (AB279654)

False colour image of Western blot : Anti-HIF-1 alpha antibody [54/HIF-1a] staining at 1/2000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab279654 was shown to bind specifically to HIF-1 alpha. A band was observed at 110 kDa in treated wild-type HCT 116 cell lysates with no signal observed at this size in HIF1A knockout cell line ab255394 (knockout cell lysate ab263860). 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-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

All lanes:

Western blot - Anti-HIF-1 alpha antibody [54/HIF-1a] (ab279654) at 1/2000 dilution

Lane 1:

Wild-type HCT 116 Untreated DMOG Control cell lysate at 20 µg

Lane 2:

Wild-type HCT 116 Treated DMOG (1 mM, 4 h) cell lysate at 20 µg

Lane 3:

HIF1A knockout HCT 116 Untreated DMOG Control cell lysate at 20 µg

Lanes 3 - 4:

Western blot - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-hif1a-hif-1-alpha-knockout-hct116-cell-line-ab255394'>ab255394</a>)

Lane 4:

HIF1A knockout HCT 116 Treated DMOG (1 mM, 4 h) cell lysate at 20 µg

Predicted band size: 92 kDa

false

• WB

Supplier Data

Western blot - Anti-HIF-1 alpha antibody [54/HIF-1a] (AB279654)

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

The expression profile/molecular weight observed is consistent with what has been described in the literature (PMID : 9159130).

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-HIF-1 alpha antibody [54/HIF-1a] (ab279654) at 1/2000 dilution

Lane 1:

Untreated HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

HeLa treated with 1 mM DFO (Deferoxamine mesylate) for 24 hours whole cell lysate at 20 µg

Lane 3:

Untreated HeLa whole cell lysate at 20 µg

Lane 4:

HeLa treated with 0.5 mM CoCl2 for 6 hours whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/50000 dilution

Predicted band size: 92 kDa

Observed band size: 110 kDa

false