SpecificityThe expected molecular weight of FIH is 40 kDa. A band corresponding to this size is seen on the western blot analysis, and is competed away by the addition of FIH blocking peptide. A smaller band is seen, which is also competed away by the blocking peptide. In the A431 extract a nonspecific band of approximately 50 kDa is seen, which is not diminished by the addition of blocking peptide.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application notesICC/IF: Use at a concentration of 5 µg/ml.
WB: 1/500. Detects a band of approximately 40 kDa (predicted molecular weight: 40 kDa). Can be blocked with FIH peptide (ab12306).
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
FunctionHydroxylates HIF-1 alpha at 'Asp-803' in the C-terminal transactivation domain (CAD). Functions as an oxygen sensor and, under normoxic conditions, the hydroxylation prevents interaction of HIF-1 with transcriptional coactivators including Cbp/p300-interacting transactivator. Involved in transcriptional repression through interaction with HIF1A, VHL and histone deacetylases.
All lanes : Anti-HIF1AN antibody (ab12289) at 1/500 dilution
Lane 1 : Hela nuclear extract Lane 2 : Hela whole cell lysate Lane 3 : A431 whole cell lysate Lane 4 : Jurkat whole cell lysate Lane 5 : Hek293 cell lysate Lane 6 : Hela nuclear extract with FIH blocking peptide at 1 µg/ml Lane 7 : Hela whole cell lysate Lane 8 : A431 whole cell lysate with FIH blocking peptide at 1 µg/ml Lane 9 : Jurkat whole cell lysate with FIH blocking peptide at 1 µg/ml Lane 10 : Hek293 whole cell lysate with FIH blocking peptide at 1 µg/ml
Lysates/proteins at 20 µg per lane.
Secondary Goat polyclonal to rabbit IgG H&L (HRP) at 1/5000 dilution
Predicted band size : 40 kDa Observed band size : 40 kDa
ICC/IF image of ab12289 stained human Hek293 cells. The cells were 4% PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab12289, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).
References for Anti-HIF1AN antibody (ab12289)
This product has been referenced in:
Zolk O et al. Activation of negative regulators of the hypoxia-inducible factor (HIF) pathway in human end-stage heart failure. Biochem Biophys Res Commun376:315-20 (2008).
Read more (PubMed: 18782560) »
Zheng X et al. Interaction with factor inhibiting HIF-1 defines an additional mode of cross-coupling between the Notch and hypoxia signaling pathways. Proc Natl Acad Sci U S A105:3368-73 (2008).
Read more (PubMed: 18299578) »