Hypoxia Antibody FAQ's
Why do I see a band at 70-85kDa in my HIF1a western blot?
Although this band has been well-documented in many labs in addition to our own, it has not been fully characterized. We believe that it is a degradation or cleavage product of HIF1a that is commonly seen in samples that have experienced protein degradation or multiple freeze-thaw cycles.
How can I get rid of this band?
It is very important to reduce this band because it can compete antibody away from the actual target, making it appear as though HIF1a is absent in your samples. It is critical to exercise the utmost care in sample preparation in order to avoid any degradation, and to use fresh samples whenever possible. It is also common practice to cut the membrane at 85 kDa in order to completely eliminate this protein from your blot; doing so also adds the benefit of not having to strip your membrane before probing for a loading control.
What does CoCl2 treatment do?
Cobalt Chloride is a useful treatment to induce hypoxic like conditions in the cells. This treatment is especially useful in positive controls to induce HIF expression. HIF expression has been shown to be affected both in a time and dose dependent manner when cells are exposed to CoCl2. Additional information on this process can be found at the following paper. “The Role of Hypoxia Inducible Factor 1α in Cobalt Chloride Induced Cell Death in Mouse Embryonic Fibroblasts” Toxicol. Sci. (2004)
What can I use as a positive control?
What can I use as a negative control?
Negative controls are recommended for our hypoxia antibodies by the lab. This is due to the fact that since several degradation products show up on blots for hypoxia antibodies, it can often make interpretation of results difficult.
If there is an available blocking peptide for your particular catalog number the lab recommends running a negative control competition assay. This will block signal of your antibody for all specific bands leaving behind only non-specific bands.
If no blocking peptide is available you may run normoxic samples in tandem with hypoxic samples to observe differences between them and how targets such as Hif degrade in the presence of oxygen.
Where will my signal be in an immunostaining technique such as IHC or ICC?
HIF-1 alpha can be found at very low levels in the cytoplasm under normoxic conditions. During hypoxia, HIF1a is stabilized and translocates to the nucleus to act as a transcription factor.You should expect to see nuclear staining in your hypoxic samples, but may observe faint cytoplasmic staining in relation to degraded HIF in your samples.
HIF-2 alpha is strongly expressed in the nucleus under hypoxic conditions.
VEGF strongly stains the cytoplasm with additional staining noted on the plasma or nuclear membrane. Spliced variants of VEGF can be secreted.
My signal is really weak, how can I make it stronger?
Your signal may be weak due to a greater amount of degraded HIF in your samples than non degraded protein. This can be alleviated by using nuclear extracts instead of whole cell extracts. Since HIF localizes to the nucleus in hypoxic conditions the signal will be enhanced as there will be less degraded HIF in nuclear extracts.
Another way to enhance your signal in Western Blot is to cut the membrane. This should enhance the signal by removing degradation products that may be competing away signal of your antibody from the non degraded protein.
In immunostaining applications ensure that you are using the appropriate permeabilizing agent to achieve the best staining. For nuclear targets a stronger detergent is often necessary and the lab will recommend Triton X-100 over Tween 20.