Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] - BSA and Azide free (AB308638)

This data was developed using ab308637, the same antibody clone in a different buffer formulation. Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HCT-116 (human colorectal carcinoma epithelial cell, Red) treated with 10µM MG-132 for 4h / Untreated HCT-116 (Green) cells labeling HIF-1 alpha (hydroxyl P564) with ab308637 at 1/50 dilution (1 ug)/Red and Green (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] - BSA and Azide free (AB308638)

This data was developed using ab308637, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HCT 116 (human colorectal carcinoma epithelial cell) cells labeling HIF-1 alpha (hydroxyl P564) with ab308637 at 1/50 (10.32 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing nuclear and weak cytoplasmic staining in HCT 116 cells after treatment with MG-132 (10 µM) for 4 hours and showing no staining in untreated HCT 116 cells. The image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Dot

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Dot Blot - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] - BSA and Azide free (AB308638)

This data was developed using ab308637, the same antibody clone in a different buffer formulation. Dot blot analysis of HIF-1 alpha (hydroxyl P564) using ab308637 at 1 : 1000 (0.516 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution. Exposure time : 15 seconds Blocking and diluting buffer and concentration : 5% NFDM/TBST

• WB

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Western blot - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] - BSA and Azide free (AB308638)

This data was developed using ab308637, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST This antibody does not cross-react with human HIFIA (hydroxyl P402) . In Western blot, Anti-HIF-1 alpha antibody (ab179483) as pan control staining at 1/1000 dilution. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 15 seconds

Lane 1:

293T cells transfected with an empty vector containi a His-tag whole cell lysate

Lane 2:

293T cells transfected with an empty vector containi a His-tag, treated with 10 uM MG-132 for 4 hours whole cell lysate

Lane 3:

293T cells transfected with a human HIFIA(WT) expression vector containi a His-tag whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a human HIFIA(WT) expression vector containi a His-tag, treated with 10 uM MG-132 for 4 hours whole cell lysate at 20 µg

Lane 5:

293T cells transfected with a human HIFIA P564G(mutant) expression vector containi a His-tag whole cell lysate

Lane 6:

293T cells transfected with a human HIFIA P564G(mutant) expression vector containi a His-tag, treated with 10 uM MG-132 for 4 hours whole cell lysate

Lane 7:

293T cells transfected with a human HIFIA P402G(mutant) expression vector containi a His-tag whole cell lysate at 20 µg

Lane 8:

293T cells transfected with a human HIFIA P402G(mutant) expression vector containi a His-tag, treated with 10 uM MG-132 for 4 hours whole cell lysate at 20 µg

false

Exposure time: 15s