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Fusion protein corresponding to Human HIF-1-alpha aa 432-528.
Under normoxic conditions HIF-1 alpha has a short half-life. It is largely undetectable in cells or tissues grown under normoxic conditions. It is stabilized only at O2 concentrations below 5% and upon stabilization under hypoxic conditions HIF-1 translocates to the nucleus. Therefore we recommend western blots using nuclear extracts and running Hypoxia treated samples as positive control (ab180880). Hypoxia can be induced with treatment using certain agents e.g. CoCl2 or DFO, etc. so proper sample preparation is critical.
Our Abpromise guarantee covers the use of ab2185 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/500 - 1/1000.|
|IHC-Fr||1/10 - 1/2000.|
|ICC||Use at an assay dependent concentration.|
|ICC/IF||1/10 - 1/2000.|
|EMSA||1/1 - 1/100.|
|ChIP||Use 25µl for 106 cells.|
ICC/IF image of ab2185 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2185, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling HIF-1-alpha with ab2185.
ChIP analysis of HIF-1-alpha genomic sequences from HeLa cells cultivated in normoxic (N) or hypoxic (Hx) conditions, using a HIF1-alpha polyclonal antibody (ab2185). For a negative control, IgG was used and the input as a positive control in the subsequent PCR. Primers for known target genes were used HIF1 alpha, A. EPO and B. VEGF.
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