Hi,

Have you tried different antibodies? Could the antibody be identifying an unspecific protein besides HIF-1 alpha? 

Hi, I do not have experience with AML cells… but I worked on HIF1a for quite some time using HeLa, 293T and RCC4 cell lines most of the time. Of course there are many possible reasons why you might be getting these results. But something that seems very unusual here is that you get such a strong staining without treatment and after a very very long CoCl2 treatment no increase. My first thought is you are getting an unspecific signal here… I had these kind of problems too and can only recommend to test a variety of antibodies before you get started with the actual experiments. Back in 2011 I tested 6 different HIF-1a antibodies… and only 2 really gave me a clean result, others were very unspecific.. Its not a properly labeled figure, but you can see the differences in results… The HIF-1a band is marked with the red arrow, and appears at 110 kDa. For the test I used HeLa cels either as control without treatment, treated 5h with 200 µM CoCl2 (every 2nd lane), and treated 3h with 20µM MG132 (every 3rd lane). As expected, CoCl2 treatment causes HIF to accumulate in its non-ubiquitinated form at about 110kDa, MG132 treatment causes HIF to accumulate at MWs higher than 110 kDa, as polyubiquitinated HIF-1a species. 

The two antibodies I used for subsequent experiments were No1, and No6: 

- BD Biosciences, HIF-1alpha, mouse monoclonal CatNo 610959, dilution 1:1000

- Novus, HIF-1alpha, rabbit polyclonal, CatNo NB100-449, dilution 1:500

Other than that I can also recommend a shorter treatment with CoCl2, 3-5h is most definitely enough. 

HIF-1a can be a pain in the neck! Good luck

Thank you Petricia and Kurt...I am totally agree with you, I think I need to try different AB before interpreting my results. the pictures I attached show that I also got strong splitted bands by WB in a region smaller than the expected MW for HIF1alpha which might also be due to non-specific binding, however, surprisingly I got no any bands in the 120 MW! Regarding the duration of CoCl2 treatment, most of the published WB results are ranging between 18 hr.s-72 h.s. in addition my model required prolonged treatment but with low CoCl2 concentration (25uM). I am not sure how much this low conc. will make a detectable increase in HIF1alpha protein by WB or IF techniques? Thanks again

Looking at your blots, I am pretty sure you are not looking at HIF1a there. The two ABs I named above should help. Also make sure you are very careful and quick with your lysis, always on ice of course… regular RIPA buffer gave me OK results. But to detect HIF1a in WB there was no better method for me than direct lysis in SDS sample buffer… take a 250 µl pre-heated 2x SDS sample buffer per well of a 6-well plate, drop it directly on your cells after washing off medium, scratch the cells from the plate, resuspend via pipet and boil for 10-15 min at 100C, then ultrasound treatment to be sure all membranes are disrupted, ready to go. 

I guess 25µM CoCl2 should work too.. I attached another file when I did my first titration experiments with 200µM CoCl2, after one hour HIF1a accumulated. 

Also look at the different lysis methods… Sample buffer delivered surprisingly clean results (also used for no 9, CoCl2 time course.. attached above), and you can't lyse your cells any quicker.  You can also see, the longer you lyse, the more HIF1a is going to be degraded within the lysis buffer… Adding CoCl2, MLN and MG132 to the lysis buffer prevents this degradation during lysis (of course you don't need to add these reagents to 2x Sample buffer). Have fun playing around! 

Hello as you guys enough knowledge about HIF 1a. can you please specify the difference in the protein expression in normal cell line and cancer cell line? how much overexpressed ?