Anti-HIF-2-alpha antibody [ep190b] (ab8365)

I was wondering, did you try any other antigen retrieval step, such as Tris EDTA (pH 8)? This has been successfully used by another researcher with human breast cancer tissue. I would suggest trying this.

I am not sure how long you incubated with the primary antibody, but if overnight incubation has not yet been tried, I would suggest trying this as well.

Thank you for contacting us.

We guarantee ab8365 to work in Flow Cyt, ELISA, IHC-Fr, IHC-P, WB with rat and human samples in accordance with our Abpromise. If you have difficulty in these applications and species, let us know and we will refund or replace the product for you within 6 months of purchase.

https://www.abcam.com/index.html?pageconfig=resource&rid=10372&pid=10000&source=pagetrap

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for your patience whilst I have been trying to contact our supplier to get the protocol they used to test ab8365 in IHC-Fr. Unfortunately they are unable to provide that information, therefore I think the best course of action is to either replace or refund the antibody for you.

Please let me know how you would you like to continue and if you could also provide either the Abcam order number or the PO# used to purchase the antibody that would be very helpful.

I look forward to your reply and I do apologize for the delay in responding to you.

Thank you for your enquiry and your interest in our products.

I can confirm that this antibody (ab8365: Anti-HIF2 alpha antibody) has been tested for IHC-Fr, IHC-P, WB, please take a look at the datasheet under tested application and the images.



ab8365 can detect endogenous HIF2 alpha, however under hypoxia HIF2 alpha is induced and the signal for HIF2 alpha in Western blot is significantly stronger compared to normoxic conditions.

If you need any further assistance in the future, please do not hesitate to contact me.

Thank you very much for your answer. I am sorry, I think I am also a little bit confused with the HIF proteins. There are indeed different studies - on particular study showing that in MEFs (i have not found yet a study on HEK cells) the localisation of HIF 2 alpha is mainly cytoplasmic and not inducible. http://mcb.asm.org/cgi/content/full/23/14/4959?view=long&pmid=12832481 The publication here below shows the HIF 2 alpha expression in liver, which seems to be also quite a lot of cytoplasmic staining. http://www.wjgnet.com/1007-9327/full/v16/i3/WJG-16-281-g001.htm Please have a look at these publications (and maybe others, as I am not a specialist with HIF 2 alpha) and let me know whether these might explain the stainings. I have also looked through the protocol provided. It seems very good to me. Can you just confirm what kind of controls you have performed to exclude unspecific staining? Have you performed an isotype control? This would indeed be the best control. I am looking forward to hear back from you with your comments.

Thank you for taking time to contact us. I am sorry to hear that this antibody is not providing satisfactory results. Having reviewed this case, I would like to offer some suggestions to help optimize the results with this antibody. Indeed, HIF 2 alpha is a difficult target to work with. I suspect that the observed staining is indeed the real staining. Upon stabilization HIF translocates to the nucleus. Please also note that Hif-2 alpha is extremely rapidly degraded. Upon cellular re-oxygenation its is completely degraded in less than 1 minute. Please see also the image of the publication, where MEFs have been stained for HIF 2 alpha and substantial cytoplasmic staining is observed. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC162224/?tool=pubmed Additionally, HIF can be difficult to detect in cells or tissues grown under normoxic conditions. Its is stabilized only at O2 concentrations below 5% or with treatment using certain agents (CoCl2, DFO, etc.) so proper sample preparation is critical. Can you please specify what hypoxic conditions have been used to treat the HEK cells? I am looking forward to hear back from you with your comments on the above raised points. I have also attached a questionnaire, which I would be grateful if you could fill it out, so I could look into the problem in more detail. Can you please also attach an image of the kidney staining? Thank you very much! In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund. I hope this information is helpful, and I thank you for your cooperation.