Did you use other tissues as control?

If you did, what was the result with the controls?

Do you use a hypoxia incubator or hypoxia mimicking drugs (e.g. CoCl2). If you have the cells in a hypoxia incubator you have to be extremely rapid for the protein isolation when you take them out as Hif1 degradation is very quick. Your 02 levels might also be still to high or simply the antibody might not work. What do you use as positive control?

Controls are key as most antibodies aren't great.

Positive controls of cells treated with cobalt chloride, nickel chloride  or DMOG are a good ways to determine if your chamber is working ( sometimes the sensors are not calibrated) and will help confirm that your cells can induce HIF (to detectable levels) .   

Lysis in hypoxic conditions also important as has been already said; it's very unstable. You can also qPCR target genes to show inducibility.

Hi Neha,

Here are some suggestions:

1- Lyse your cells very rapidly on ice, within a few minutes, since HIF1A degrades quickly

2- Maximize the amount of protein you're loading

3- Use adequate protease inhibitors

4- Some recommend using nuclear extracts instead of whole cell extract

I have used U2OS cells treated with hypoxia as positive control, even the positive control did not show HIF band on the blot. We use the chamber, and it is the big chamber. We trypsinize the cells and bring them out.

Thank you all for your suggestions. I will look forward to more.

Try CoCl2 as positive control. If this is also not working, you will need a different antibody.