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Fusion protein corresponding to Human HIF-1-alpha aa 400-550.
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).
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 DMSO, 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 DyLight 488 goat anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in 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.
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 (blue) and DFO treated (red) cell traces are shown.
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