I have been trying to study the expression of HIF 1 alpha under hypoxia conditions in NSCLC cell lines. I incubate the cells in small hypoxia incubators (brincubators) and flush them 1% oxygen gas. I monitor hypoxia levels in the incubator by using oxygen sensors. While lysing the cells, I put the brincubator in an atmos bag and flush it with 1% oxygen so that the cells are still hypoxia when I lyse them for western blots and PCR. But, I still don't see HIF1 alpha expression. I have tried different antibodies, Cocl2 treatment using different cell lines (HeLa, EAhy926 etc) but no positive results so far. Does anyone have nay suggestions for this?
It would be worth trying a shorter time period such as 24 or 48hr as it has been suggested that down-regulation of HIF-1a occurs during prolonged hypoxia through a negative feedback loop.
another thing worth trying is keeping the samples on ice - in my experience if you transfer the cells on ice before rinse/lyse, I could detect HIF1 no problem, and that's either collection within a hypoxic chamber, or outside (although outside will need to be collected in less than a minute)
Been a long time since I've done this, but if you are not getting any HIF expression using multiple cell lines and with CoCl2 this suggests something a bit more fundamental. I agree that 4 days is not needed and possibly too long. You should see it within a few hours. You could also try DMOG as another positive control.
All great suggestions. If these don't work you may need to test it on nuclear lysates from these cells. Also use a tested positive control.
Thanks for your suggestions. I am planning to try acute hypoxia conditions and also CoCl2.
HIF1A is among the most rapidly degrading proteins and it is a very common issue that researchers face during its WB analysis. Degradation results in very weak specific band (~110kDa) and/or multiple degradation bands (between 40-80kDa). Key to deal with this issue is to prepare the lysates as quickly as possible (on ice/at 4˚C) after the collection of cells/tissues, preferably in a hypoxic chamber. Also, true hypoxia or hypoxia mimetic based positive controls (e.g. cells treated with CoCl2, DMOG etc) are helpful to figure out the root cause of issue or for differentiating the specific band.
For additional troubleshooting tips and more than 20 most frequently asked questions / FAQs on Hypoxia/HIFs, I would suggest reading: http://www.novusbio.com/support/hypoxia-and-hif-faqs
Hello Everyone. Thanks for your suggestions. As suggested by Shing-hu Cheung, I tried acute hypoxic conditions. When I first treated my cells under chronic hypoxia, I saw a significant decrease in HIF-1a levels at the mRNA level and I couldn't see any expression of HIF-1a at the protein level. Later, I changed the antibody and I tried acute hypoxic conditions. I noticed that the cell line I am using (NCI-H292) expresses HIF-1a under normoxic conditions as well. However, acute hypoxia did not affect HIF-1a expression at the mRNA or protein level. I did test for the specificity of the antibody I am currenlty using.
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