I am trying to do western blotting of tumor samples for HIF-1a. The antibody is from neomarkers and I used a dilution of 1:1000. The protein samples from the tumor were isolated in RIPA buffer. I tried several times, but could not get the specified band.
Any help would be highly appreciated.
hi luana
Thnx for your answer.
Yes, I tried this antibody with CoCl2 treated HUVEC cells (positive cont.); however no band was seen at 120 KD (expected size). I just saw a faint band around 57 kD,
In tumor samples, i can just see a smear.. no specific band though.
How much protein(ug) r u loading ?, it is better to load high amount of protein if ur not getting for less amount of protein. 2nd thing you can reduce the dilution1:500 .3rd wether loading control is good like actin/gapdh/tubulin?, incubate antibody(primary) overnight in 4degree shaker that will be goood.....
Hi Vijayendra,
I agree with Luana about the antibody choice. Previously, I had tried to blot for HIF-1a with an Abcam antibody and was getting a few bands of smaller size and nothing at ~120 kDa. As soon as I switched to the BD antibody that Luana has suggested I got nice bands in HeLa cells treated O/N with CoCl2 150uM. Personally, I have not yet blotted for HIF-1a from tissue so this may generate other concerns that I am unfamiliar with. Good Luck!
Luana:
I used 100 uM CoCl2 for 24 h incubation. the change of antibody indeed seems a good idea. I have faced similar problems with other antibodies from different companies. I will try that. Thnx.
Santosh: its 30 ug of protein. there is no problem with actin... its giving quite good band. I already tried overnight 4 degree incubation.... didnot work :(
Sean: Thnx sean! I will try with different antibody.
Hi Luana
Does the antibody u suggested "Mouse Anti-Human HIF-1α, Cat. #: 610958 from BD Transduction Laboratories." work for mouse samples too?
Hi Vijayendra,
We have had consistent success using anti-HIF-1a antibody mentioned by Luana. This is the NB100-479 from Novusbio.
I strongly recommend that you include a positive control in your Western blots. For positive control, we routinely used lysates from COS7 cells treated with 400uM CoCl2 for 4 hours; also include lysate from untreated cells to confirm specificity of the induction. You should get a non-specific immunoreactive band around 120kDa, and a specific induced band just below that.
Good luck.
HI Vijayendra,
If positive controls work (CoCL2 treated cells is indeed a good one), It might be because HIF-1a is degraded in your samples (half live less than 3 minutes). If it is indeed degraded, you can include proteasome inhibitors in your extraction (and perfusion if mouse tissues). If samples are not easy to get, you may want to use cabonic anhydrase 9. It is much more stable and in our hands a very robust marker of hypoxia.
Good luck!
Hi Vijayendra,
In case you are still looking for help, I've found this HIF1alpha antibody from StressMarq Biosciences (Catalog# SMC-184) that might work for you. It is reactive in human, mouse, rat, and cow tissues. It has been tested for use in western blot using HeLa cell lysates at a 1:500 dilution. They also offer a free trial program, so you could test it out before you buy it. Hope this helps!
http://www.stressmarq.com/Products/Antibodies/SMC-184D.aspx
hi I have a Question: am trying to blot HIF 1 alpha in MCF 7 cells, but unfortunately i don,t have a hypoxia chamber. Can any one suggest some alternative inexpensive & accurate method to maintain hypoxia condition in CO2 incubator.
Hi Venkat,
It could be difficult and a bit expensive to work hypoxic conditions using a standard incubator, especially if you don,t have a way to acurately measure O2 concentrations. I would suggest using CoCL2 to stabilize HIF-1alpha in your cells.
Good luck!
Claire
Thank You Clarie
Can you send me the procedure using CoCl2 to stabilize HIF 1 alpha cells
Just check this protocol, can we follow this?
Nat Protoc. 2006;1(4):2088-90.
Inexpensive low-oxygen incubators.
Wright WE1, Shay JW.
Abstract
Although the evidence is overwhelming that ambient oxygen is at least somewhat damaging to most normal cells in culture, the expense and effort involved has resulted in few laboratories growing their cells under physiological oxygen conditions. We here describe how to produce, from commercially available plastic wide-mouth jars, very simple gas-tight containers that can be flushed with prepared gas mixtures to produce low-oxygen environments for standard cell culture. This permits any laboratory to easily try the effects of physiological oxygen on their system without the need for dedicated incubators and substantial monetary investments.
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