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Fusion protein corresponding to Human HIF-1 alpha aa 400-550.
This antibody clone is manufactured by Abcam.
HIF-1 alpha can be a difficult target to work with so we have compiled a summary of all the important information you need to know including useful tips. This can be found in the protocols tab or alternatively click here to download it.
Western blot protocol advice:
For best results in Western blot using this antibody, we recommend the following:
1) Using positive control samples such as DFO or CoCl2 treated cell lysates.
2) Ensure cell lysis occurs quickly (within 2 mins) if removed from hypoxia.
3) Use 4-12% or 10% Bis-Tris precast gels. Run at 200 V for 50 mins. MOPS buffer.
4) Nitrocellulose membrane. Transfer at 30 V for 70 mins.
5) Blocking: 5% milk in TBST (0.05% Tween). 1 hour at room temperature.
6) Primary antibody: use ab1 at 5 µg/mL. Dilute in 2% milk in TBST (0.05% Tween). Incubate for 2 hours at room temperature.
7) Washing: TBST (0.05% Tween) for 8 minutes (repeat 3 times).
8) Secondary antibody: dilute in 2% milk in TBST (0.05% Tween). Incubate for 90 mins at room temperature.
Our Scientific Support team will be happy to provide further information and advice.
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.
ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
|ICC/IF||1/100 - 1/200.
|WB||Use a concentration of 5 µg/ml. Detects a band of approximately 120 kDa (predicted molecular weight: 92 kDa).
We recommend blocking for 1 hour with 5% milk in TBST and reducing to 2% milk in TBST for the primary and secondary antibody incubation steps. For primary antibody incubation, we recommend 2 hours at room temperature.
We recommend using 5% milk in TBST as the blocking agent, decreasing to 2% milk in TBST during primary and secondary antibody incubation.
Blots were developed with Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) secondary antibody
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.
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.
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).
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.
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.
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