I am working on 3D tumors expected to have intrinsic hypoxia. For protein isolation, I wash my samples with PBS and use RIPA buffer with protease inhibitors within a 5-minute time frame, as HIF1α is rapidly degraded. However, I have not been able to detect HIF1α in my western blots.

I also tried fixing my samples with 4% PFA for 20 minutes, followed by both permeabilization and no permeabilization, and then adding RIPA buffer with protease inhibitors, but my BCA protein estimation shows no detectable color change compared to the blank.

What could be causing this issue, and how can I improve my protocol? If I switch to adding Laemmli buffer directly and boiling the samples before protein isolation, how should I quantify the protein? I am using the HIF1α antibody from Cell Signaling Technology. Any guidance would be greatly appreciated!