All lanes : Anti-HIF1 beta antibody [EPR17516] (ab184711) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : ARNT knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 87 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab184711 observed at 90 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab184711 was shown to react with ARNT in HAP1 wild-type cells in Western blot. Loss of signal was observed when ARNT knockout sample was used. HAP1 wild-type and ARNT knockout whole cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween^®) before incubation with ab184711 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 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 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded Human uterus tissue labeling HIF1 beta with ab184711 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on epithelial cells of Human uterus is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human hepatocellular carcinoma tissue labeling HIF1 beta with ab184711 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on cancer cells of hepatocellular carcinoma is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Anti-HIF1 beta antibody [EPR17516] (ab184711) at 1/1000 dilution + Human fetal kidney lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 87 kDa
Observed band size: 87 kDa


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-HIF1 beta antibody [EPR17516] (ab184711) at 1/1000 dilution + HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 10 µg

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

Predicted band size: 87 kDa
Observed band size: 87 kDa


Exposure time: 8 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-HIF1 beta antibody [EPR17516] (ab184711) at 1/2000 dilution

Lane 1 : Human pancreas lysate
Lane 2 : Human colon lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 87 kDa
Observed band size: 87 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.