I am doing HIF1 westerns in southwestern HIF overexpressing mice liver and adipose tissue. I'm using novus antirabbit HIF1a antibody 1:1000 concentration overnight incubation and getting strong bands around the 90kD area. Now, HIF MW is 93kD, but in westerns, the HIF band is usually around 120kD according to the internet. Am I getting the right HIF band or just some non specefic bands? Any help is appreciated..
Thanks a lot
This papers could help
http://www.ncbi.nlm.nih.gov/pubmed/20458283
http://www.ncbi.nlm.nih.gov/pubmed/19840250
Dileep,
As I'm sure you know, working with HIF can be very tricky. I honestly had no luck with the Novus antibody, it gave me way too many non-specific bands for human cell lines. So far, I've found that Cayman is the best for mouse tissue and Millipore or BD are best for human cells.
HIF-1a from mouse mast cells gives us a band at around 130 KDa with the Cayman ab, this is after incubation of cells at 1% oxygen. It could be that you are getting a partially degraded product. What does your isotype control look like? If I'm not mistaken, it should be mouse IgG2b for the Novus. Also 2 things that really helped me with my WB are:
1) adding more protease inhibitors to my lysis/homogenization buffers
2) cutting the membrane along the 90-95 KDa line before exposing, so that any fainter bands that may have been masked before by the fat non-specific bands will not appear clearly.
Hope I was able to help, best of luck!
Thanks a lot Nathalie, helped a lot. Could you also tell me if you were using gradient gel?
You're very welcome Dileep. I was in fact using a 10% SDS gel, nothing special. I use 10% because I also blot for RPL13A, a housekeeping prot that is not affected by hypoxia (GAPDH and Actin and upregulated by HIF) and its at 20KDa, so a 10% gel allows me to look at both proteins on the same membrane. You could however use a smaller % gel, like 8 or 6 if you want a clearer breakdown of the different HIF bands.
Hi Dileep,
We're routinely doing HIF1 and HIF2 immunoblotting on Westerns with the Novus antibodies on cultured cells from mouse brains. We run the cos-7 hypoxia-induced lysate as the positive control (as suggested). We routinely see bands around the 110-120kDa mark that correspond to the bad on the positive control. This is with both whole cell and/or nuclear lysates. we also see many other bands above it but since we also blot for other proteins that are around the 60kDa, we usually cut around the 80-90kDa mark so i can't confirm if we see those bands unfortunately :( But, we've been in touch with Novus regularly about this, and they say we have the right band. Mybe it is a tissue (brian vs adipose) thing? They did suggest to run nuclear lysates for more clarity, use 1% milk to reduce background (works!) . I agree with Nathalie about cutting around 90 so that you're making the fainter bands clearer..
Thanks a lot Praneeti, we are planning to do HIF westerns in mouse brain as well. Is the nuclear extraction protocol any different from the other tissue? Do you perfuse mouse with PBS before harvesting the brain? Is there any thing to keep in mind to get the best results?
Thank again
Dileep
Hi Dileep,
Apologies for my late reply!
We use the Nuclear/Cyto kit from Pierce Biotech (http://www.piercenet.com/product/ne-per-nuclear-protein-extraction-kit) so I'm assuming it is the same as any tissue. All my protein is from cultured cells and i do wash them twice with cold PBS before starting extraction. The only thing i can think of to suggest is that you make sure that your cells/tissue is lysed properly. I have adherent cells that i scrape off (as I'm not keen on using trypsin to lyse them) and my nuclear protein yields are usually really low.. I can imagine that might be an issue with tissue too perhaps?
Are you doing specific areas of the brain or whole cortex? I ask as someone in my lab tried to do whole brain lysates to look at HIF1 but didn't detect much .. we use unilateral injury model and the point was to compare the injured to uninjured side but we didn't see much...
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