Thread: HIF1alpha western blot

Hi,

I really need help with my HIF1alpha western blot. I'm trying to detect HIF1alpha in mouse heart essentially but also in rat neonatal cardiac myocytes. I have used several different conditions: PVDF membranes, nitrocellulose, unboiled samples, boiled ones, antibodies from Santa Cruz and from R&D which are used in the litterature. I treated cells with CoCl2 that is an HIF1a stabilizer. Nothing works for me. Please, help me with any suggestions. Thank you.ss

We use R&D's antibody and seems to work fine. We also use PVDF, heat samples to 60 (not boil) for 10 mins. we have used CoCl2 and our hypoxia chamber and seen similar results for both.

The only other thing I can think about is the way you collect protein. But I guess if you are using CoCl2 it doesn't matter how fast you are at isolating the protein. You can always contact the antibody suppliers.