All lanes : Anti-HIF-1 alpha antibody [EPR16897] (ab179483) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Dimethyloxaloylglycine, Prolyl hydroxylase (PHD) inhibitor (ab141586)
Lane 3 : HIF1A knockout HAP1 whole cell lysate
Lane 4 : HIF1A knockout HAP1 treated with DMOG (0.5mM 18hr) whole cell lysate
Lane 5 : HeLa whole cell lysate
Lane 6 : HeLa treated with DMOG (0.5mM 18hr) whole cell lysate

Lysates/proteins at 40 µg per lane.

Predicted band size: 92 kDa
Observed band size: 105 kDa

why is the actual band size different from the predicted?

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).

All lanes : Anti-HIF-1 alpha antibody [EPR16897] (ab179483) at 1/5000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) treated with 0.5 mM CoCl2 for 6 hours whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 92 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

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).

All lanes : Anti-HIF-1 alpha antibody [EPR16897] (ab179483) at 0.163 µg/ml

Lane 1 : Untreated C6 (rat glial tumor glial cell), whole cell lysate
Lane 2 : C6 treated with 400 µM CoCl2 and 200 µM MG-132 (ab141003) for 4 hours

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 92 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?


Exposure time: 26 seconds

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

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) cell lysate treated with 0.5mM CoCl2 (Cobalt(II) chloride) for 6 hours.
Lane 2 : Untreated HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 92 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-HIF-1 alpha antibody [EPR16897] (ab179483) at 1/1000 dilution

Lane 1 : NIH/3T3 (Mouse embyro fibroblast cells) cell lysate treated with 0.1mM CoCl2 for 24 hours.
Lane 2 : untreated NIH/3T3 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 92 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.