Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

HIF-1 alpha was immunoprecipitated using 0.5 mg HeLa Nuclear DFO treated whole cell extract (ab180880), 5 µg of Mouse monoclonal to HIF-1 alpha and 50 µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10 minutes, HeLa DFO treated whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10 minutes under agitation.

Proteins were eluted by addition of 40 µl SDS loading buffer and incubated for 10 minutes at 70°C; 10 µl 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: 110 kDa; HIF1 alpha

ab1 staining HIF-1 alpha in rat infarct core striate tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/1000) for 1 hour at 25°C. An HRP-conjugated rabbit anti-mouse IgG whole molecule (1/200) was used as the secondary antibody. 

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ab1 staining HIF-1 alpha in mouse skin tissue sections by Immunohistochemistry. Tissue was fixed with formaldehyde and blocked with 10% serum for 30 minutes at 22°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/100) for 1 hour at 25°C. An HRP-conjugated Goat anti-mouse was used as the secondary antibody.

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ab1 staining HIF-1-alpha in MCF7 (Human breast adenocarcinoma cell line) cells treated with metformin hydrochloride ab12084, 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 24 hours 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 2 hours 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. Goat Anti-Mouse IgG H&L (DyLight^® 488) preadsorbed (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab1 staining HIF-1 alpha in mouse liver tissue section by Immunohistochemistry (Frozen sections). Tissue samples were fixed with formaldehyde and permeabilized 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).

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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.

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Flow cytometry using ab1. HeLa (Human epithelial cell line from cervix adenocarcinoma) 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 µg/mL). 1% BSA in PBS was used as the blocking buffer throughout. ab1 was labeled with and anti-mouse Alexa-Fluor^® 488 dye. Unstained (black), untreated (red) and DFO treated (blue) cell traces are shown.

ChIP assays were performed using ab1 to verify the binding between HIF-1α and HREs. Primer 1 (P1) and primer 2 (P2) were designed to detect HREs1 and HREs2, respectively. PCR products were separated by gel electrophoresis on 2% agarose gel.