All lanes : Anti-HIF-1 alpha antibody [H1alpha67] (ab1) at 5 µg/ml

Lane 1 : HeLa nuclear extract lysate (ab150036) at 40 µg
Lane 2 : Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (ab180880) at 40 µg
Lane 3 : HeLa nuclear control at 40 µg
Lane 4 : HeLa nuclear DFO treated at 40 µg
Lane 5 : Recombinant Human HIF-1 alpha protein (ab154478) at 0.001 µg

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 92 kDa


Exposure time: 20 minutes

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

Anti-HIF-1 alpha antibody [H1alpha67] (ab1) at 1/400 dilution + Human whole cell lysate (human lung adenocarcinoma cell line ADLC-5M2) treated for 16 hours with 100 micromolar deferoxamine (DFO) at 20 µg

Performed under reducing conditions.

Predicted band size: 92 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?



PVDF membrane was used and blocked for 16 hours in 5% milk.

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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 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.