If you are looking for WB aplications we have observed that that Ab is specific to HIF-2a using for example HepG2 shHIF2a knocdown cells. But yes, you can find a lot of inespecific bands depending on the cel line, but provided you have a good positive (e.g. hypoxia 8 hrs, 1% O2) or negative (knockdown specific to HIF2a) control you should be able to tell and convince people that you are looking at the right band.
For some cell lines it is actually fairly clean.
Look for example at figure S1, HepG2 cells, of this publication:
http://jcs.biologists.org/content/126/6/1454.full.pdf+html?with-ds=yes
You can always talk to ulrike brunning on Peter Carmellit group, they are there in leuven I think.
Good luck
Hi Gommar,
Very good question,I am also looking forward to have other researchers opinion/advices on the topic.
We use the Novus #NB100-122 for immunohistochemistry detection and are pretty satisfied of it. For western blot application we use the reference below:
goat anti mouse HIF-2a poly IgG (R&D AF2997) 1:200
It gives also few aspecific bands, but a clear signal at the expected size (positive and negative controls have been run in parallel).
I hope it will help
Christophe
Hello,
we also use the R&D antibody for HIF-2alpha in Western blot. There are only very few unspecific bands that are easily to distiguish from the HIF band. The other one that works for human HIF-2alpha in our hands is Abcam ab199. We also validated the specificity with knockdowns.
I hope it will also work for you
Best
Nathalie
Nb100-122 is Fine for human and mouse. In western It shows unspecific bands around 150 and 100 kD so the only trick you need is to separate the proteins a bit longer. Immunofluorescence and histo works as well. For chip it is kind of too dirty... You always get unspecific bands. But for western: Thinner gel and longer run. We tested it against both species with siRNA and it's perfect. Just a bit dirty ;) abcam is a lot more unspecific... I am not sure about Santa Cruz though... Good luck...
We used the NB100-122 with human samples for WB and it worked fine. The AB shows several unspecific bands but with an appropiated positive control (cells cultured under hypoxia for 4-8 hours) it will be no problem to identify the correct band.
Good luck
I have used both Novus and R and D antibody for HIF-2a for WB and immunohistochemistry in tissues. It worked fine.
Very good question.
Our own experience with both polyclonal and monoclonal antibodies anti-HIF2 from Novus and Abcam in WB is just satisfactory. Indeed we have noticed that the image differs from the type of cells. eg in human hepatocytes (Huh7, HepG2) we have many more non- specific zones and show unspecific bands around 100 and 70 kD while the image of WB with Hela cells is more clean. I am not sure about Santa Cruz.. We have not tested R and D antibody for HIF-2a.
Where we still have difficulties is to find a good anti-HIF 2 for immunofluorescence…..
Best
Panagiotis
I used the NB100-122SS for immunohistochemical analyses on human intestinal tissue and it works pretty well - I got specific stainig signals. Never tried for WB.
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