Synthetic peptide corresponding to Human HIF-1-alpha aa 1-31 (N terminal) conjugated to Keyhole Limpet Haemocyanin (KLH).
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 ab104263 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||1/10 - 1/50.|
|WB||1/50 - 1/100. Predicted molecular weight: 93 kDa.|
|IHC-P||1/10 - 1/50. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
Immunofluorescence of HeLa cells labelling HIF-1-alpha with ab104263. Hela cells were fixed with 4% PFA (20 min), permeabilized with Triton X-100 (0.1%, 10 min), then incubated with ab104263 (1:25, 1 h at 37℃). Alexa Fluor® 488 conjugated donkey anti-rabbit antibody (green) was used as the secondary antibody (1/400, 50 min at 37℃). Cytoplasmic actin was counterstained with Alexa Fluor® 555 (red) conjugated Phalloidin (7units/ml, 1 h at 37℃) and nuclei were counterstained with DAPI (blue) (10 µg/ml, 10 min). HIF1 alpha immunoreactivity is localized to the cytoplasm and nucleus.
ab104263, at a 1/50 dilution, staining HIF-1-alpha in formalin fixed and paraffin embedded lung carcinoma by by Immunohistochemistry, followed by peroxidase conjugation of the secondary antibody and DAB staining.
ab104263 has not yet been referenced specifically in any publications.