I have been doing Western blots for HIF1alpha in cancer cell lines. The predicted band size of HIF1alpha is ~116 kDa. I get a band at this size but also typically get an additional band at ~200 kDa. Just wondering if anyone has any insight into what this band is? I use reducing, denaturing conditions so I expect it to not be a dimer with HIF1beta or HIF1alpha bound to oxygen dependent degradation enzymes (e.g VHL), or is it still possible to get these dimers if their binding is strong enough? I use the antibody from BD Biosciences (http://www.bdbiosciences.com/ptProduct.jsp?prodId=29554&catyId=746071&page=product).
This situation does happen in WB assay, I used this antibody. just ignore the top band. it's no bother your result when you crop your target band.
Is this band always present (even if you use a cell lysate from other cells)? If yes, then you have unspecific bands.
If the band changes with HIF1a (for example gets smaller when HIF1a gets down), then you might see some modified HIF1a (complexes containg HIF1a are relative unlikely, since SDS-Page completely denaturates proteins).
Anyone interested in the differences in interaction of of 'cells' with HIF-1-alpha and rHuEPO? say bone marrow or K562? reticulocytes? please let us have an e-mail exchn age discussion?
We had the same problem with Cell Signaling #14179 (D2U3T) rabbit mAb antibody. The >200 kDa bands were much more intense than the ~120kDa. It may be dimers unless they are covalently linked. Publications using other HIF-1a antibodies did not have this problem.
Having the same thing with Cell Signaling #14179 (D2U3T) rabbit mAb in bone marrow derived macrophages. Exactly the same as Andrew M Chan described. It varies in my samples but does not strongly correlate with the real HIf1a band. Can it be a dimer with Hif1b?
- Badges
- Science method