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Fusion protein corresponding to Human HIF-1-alpha aa 400-550.
For Mouse specific Hif-1-alpha rabbit monoclonal antibody, please see Ab179483 (clone ID: EPR16897).
Ab179483 has been confirmed for Mouse sample in WB.
Our Abpromise guarantee covers the use of ab1 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use at an assay dependent concentration. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.|
|IP||Use a concentration of 5 µg/ml.|
|ChIP||Use at an assay dependent concentration.|
|IHC-Fr||Use at an assay dependent concentration.|
|Flow Cyt||Use at an assay dependent concentration.|
|ICC/IF||Use at an assay dependent concentration.|
|WB||Use a concentration of 5 µg/ml. Detects a band of approximately 120 kDa (predicted molecular weight: 92 kDa).
Abcam recommends using 5% milk as the blocking agent.
ab1 at 1/200 dilution staining HIF-1-alpha in human 293FT cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in formaldehyde, permeabilized in 0.5% Trition X-100 and blocked in 5% BSA for 1 hour at 25°C. The primary antibody was used at 1/200 dilution in PBS and incubated with sample at 4°C for 12 hours. An Alexa Fluor® 488 conjugated Goat polyclonal to mouse IgG was used as secondary at 1/500 dilution.
ab1 staining HIF-1-alpha in mouse liver tissue section by Immunohistochemistry (Frozen sections). Tissue samples were fixed with formaldehyde and permeablized with 0.2% Triton-X100 before blocking with 2% BSA for 30 minutes at 20°C. The sample was incubated with primary antibody (1/200) for 9 hours at 4°C. An Alexa Fluor®555-conjugated Goat polyclonal to mouse IgG was used as secondary antibody at 1/200 dilution. DAPI was used to stain the cell nuclei (blue).
Abcam recommends using 5% milk as the blocking agent, decreasing to 2% milk during primary and secondary incubation. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.
HIF-1-alpha was immunoprecipitated using 0.5mg HeLa Nuclear DFO treated whole cell extract (ab180880), 5µg of Mouse monoclonal to HIF-1-alpha and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, HeLa DFO treated whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab1.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1:20,000 dilution.
Band: 110kDa; HIF1 alpha
This image is taken from an Abreview submitted by Mike Campa, no further information is known about this imagePVDF membrane was used and blocked for 16 hours in 5% milk.
ab1 staining HIF-1-alpha in MCF7 cells treated with metformin hydrochloride (ab120847), by ICC/IF. Decrease in HIF-1-alpha expression correlates with increased concentration of metformin hydrochloride, as described in literature.
The cells were incubated at 37°C for 24h in media containing different concentrations of ab120847 (metformin hydrochloride) in water, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab1 (10 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A goat anti-mouse DyLight 488 antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.
Flow cytometry using ab1. HeLa cells were cultured untreated or with 1mM Deferoxamine (ab120727) for 24 hours to induce HIF-1-alpha protein levels. Cells were then trypsinized, fixed with paraformaldehyde and stained with ab1 (0.5 micrograms/mL). 1% BSA in PBS was used as the blocking buffer throughout. ab1 was labeled with and anti-mouse Alexa-488 dye. Unstained (black), untreated (red) and DFO treated (blue) cell traces are shown.
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