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All lanes : Anti-HIF-1-alpha [EP1215Y] antibody (TA303535) at 1/2000 dilution. Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate; Lane 2 : Hela-DFO treated (0.5mM, 24h) Nuclear Lysate. Lysate/protein at 40ug per lane. Secondary: goat anti-rabbit IgG H&L at 1/10000 dilution. Performed under reducing conditions.
HIF-1-alpha was immunoprecipitated using 0.5mg HeLa Nuclear DFO treated whole cell extract, 5ug of Rabbit polyclonal to HIF1 alpha and 50ul 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 40ul SDS loading buffer and incubated for 10min at 70°C; 10ul of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with TA303535. Secondary: Mouse monoclonal Secondary Antibody to Rabbit IgG light chain (HRP).
All lanes : Anti-HIF-1-alpha [EP1215Y] antibody (TA303535) at 1/2000 dilution. Lane 1 : HeLa Whole Cell Lysate (untreated, negative control); Lane 2 : HeLa DFO treated (0.5mM, 24h) Whole Cell Lysate; Lane 3 : HeLa Nuclear Cell Lysate (untreated, negative control); Lane 4 : HeLa Nuclear DFO treated (0.5mM, 24h) Cell Lysate. 40ug lysate/protein per lane. Secondary: goat anti-rabbit IgG H&L (HRP) at 1/10000 dilution. Performed under reducing conditions.
Anti-HIF-1-alpha antibody (TA303535) reactivity with reduced Hep3B cell lysate after transient transfection of scrambled siRNA (lanes1-3 and 7-9) or HIF-1-alpha siRNA (lanes 4-6 and 10-12). Cells were incubated at with 21% O2 (lanes 1-6) or 1% O2 (lanes 7-12) for 4h before lysis. After SDS-PAGE, membranes were blocked in 5% milk for 1h at 25°C before incubation with TA303535 (1/1,000 dilution 5% milk) for 16h at 4ºC. The blot was then incubated with an anti-Rabbit HRP-conjugated secondary antibody before developing with ECL.
Immunohistochemical analysis using TA303535 showing positive staining in Breast carcinoma tissue.
Immunohistochemical analysis using TA303535 showing positive staining in Colonic adenocarcinoma tissue.
Immunohistochemical analysis using TA303535 showing positive staining in Squamous cell cervical carcinoma tissue.
HeLa cells were untreated or treated with 1mM Deferoxamine (DFO) for 24h and fixed with paraformaldehyde for imaging by fluorescent microscopy. Cells were blocked and stained with 1X blocking buffer. TA303535 was used at 1:500. DAPI was used to label the nucleus. HIF1 alpha staining is absent in untreated cells and induced by DFO treatment. HIF1 alpha localizes to the nucleus.
TA303535 staining HIF-1-alpha in HepG2 cells treated with baicalein, by ICC/IF. Increase in HIF-1-alpha expression correlates with increased concentration of baicalein as described in literature. The cells were incubated at 37°C for 6h in media containing different concentrations of baicalein 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 TA303535 (5 ug/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight488 goat anti-rabbit polyclonal antibody at 1/250 dilution was used as the secondary antibody.
Overlay histogram showing HeLa cells stained with TA303535 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (TA303535, 1:50 dilution) for 30 min at 22C. The secondary antibody used was DyLight488 goat anti-rabbit IgG (H+L) at 1/500 dilution for 30 min at 22C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1ug/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.