Overview

  • Product name
  • Description
    Rabbit polyclonal to HIF-2-alpha
  • Host species
    Rabbit
  • Specificity
    ab20654 detects a HIF-2-alpha specific band at ~96 kDa in both unstimulated-PC12 cytoplasmic lysate and hypoxia-treated PC12 nuclear lysate. We do not know the identity of the additional bands observed. However, the bands at ~70 kDa (this could be HIF3 alpha) and 220 kDa have been observed with other HIF-2-alpha antibodies (Kiettzmann et al, 2001; Biochem. J. 354, pp531–537).
  • Tested applications
    Suitable for: WB, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Rat, Human
    Predicted to work with: Mouse
  • Immunogen

    Synthetic peptide corresponding to Mouse HIF-2-alpha aa 600-700 (internal sequence) conjugated to Keyhole Limpet Haemocyanin (KLH).
    (Peptide available as ab21447, ab21447, ab21447, ab21447)

  • Positive control
    • PC12 (Rat adrenal pheochromocytoma cell line) Cytoplasmic Lysate (ab14883) PC12 (Rat adrenal pheochromocytoma cell line) Nuclear Lysate - hypoxia treated (ab14886)

Properties

Applications

Our Abpromise guarantee covers the use of ab20654 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use a concentration of 1 µg/ml. Detects a band of approximately 96 kDa (predicted molecular weight: 96 kDa).
ICC/IF Use a concentration of 1 µg/ml.
IHC-P 1/2500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • Function
    Transcription factor involved in the induction of oxygen regulated genes. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Regulates the vascular endothelial growth factor (VEGF) expression and seems to be implicated in the development of blood vessels and the tubular system of lung. May also play a role in the formation of the endothelium that gives rise to the blood brain barrier. Potent activator of the Tie-2 tyrosine kinase expression. Activation seems to require recruitment of transcriptional coactivators such as CREBPB and probably EP300. Interaction with redox regulatory protein APEX seems to activate CTAD.
  • Tissue specificity
    Expressed in most tissues, with highest levels in placenta, lung and heart. Selectively expressed in endothelial cells.
  • Involvement in disease
    Defects in EPAS1 are the cause of erythrocytosis familial type 4 (ECYT4) [MIM:611783]. ECYT4 is an autosomal dominant disorder characterized by increased serum red blood cell mass, elevated hemoglobin concentration and hematocrit, and normal platelet and leukocyte counts.
  • Sequence similarities
    Contains 1 basic helix-loop-helix (bHLH) domain.
    Contains 1 PAC (PAS-associated C-terminal) domain.
    Contains 2 PAS (PER-ARNT-SIM) domains.
  • Post-translational
    modifications
    In normoxia, is probably hydroxylated on Pro-405 and Pro-531 by EGLN1/PHD1, EGLN2/PHD2 and/or EGLN3/PHD3. The hydroxylated prolines promote interaction with VHL, initiating rapid ubiquitination and subsequent proteasomal degradation. Under hypoxia, proline hydroxylation is impaired and ubiquitination is attenuated, resulting in stabilization.
    In normoxia, is hydroxylated on Asn-847 by HIF1AN thus probably abrogating interaction with CREBBP and EP300 and preventing transcriptional activation.
    Phosphorylated on multiple sites in the CTAD.
    The iron and 2-oxoglutarate dependent 3-hydroxylation of asparagine is (S) stereospecific within HIF CTAD domains.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • Basic helix loop helix PAS protein MOP2 antibody
    • Basic-helix-loop-helix-PAS protein MOP2 antibody
    • bHLHe73 antibody
    • Class E basic helix-loop-helix protein 73 antibody
    • ECYT4 antibody
    • Endothelial PAS domain containing protein 1 antibody
    • Endothelial pas domain protein 1 antibody
    • Endothelial PAS domain-containing protein 1 antibody
    • EPAS 1 antibody
    • EPAS-1 antibody
    • EPAS1 antibody
    • EPAS1_HUMAN antibody
    • HIF 1 alpha like factor antibody
    • HIF 2 alpha antibody
    • HIF-1-alpha-like factor antibody
    • HIF-2-alpha antibody
    • HIF2-alpha antibody
    • HIF2A antibody
    • HLF antibody
    • Hypoxia inducible factor 2 alpha antibody
    • Hypoxia inducible factor 2 alpha subunit antibody
    • Hypoxia-inducible factor 2-alpha antibody
    • Member of PAS protein 2 antibody
    • Member of pas superfamily 2 antibody
    • MOP 2 antibody
    • MOP2 antibody
    • PAS domain-containing protein 2 antibody
    • PASD2 antibody
    see all

Images

  • ICC/IF image of ab20654 stained rat PC12 cells. The cells were PFA fixed (10 min), permabilised in PBS-T (20 min) and incubated with the antibody (ab20654, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

     

  • Image courtesy of Human Protein Atlas

    ab20654 staining HIF-2-alpha in Adrenal gland. The paraffin embedded human tissue was incubated with ab20654(1:2500 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. Ab20654 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines.

    Further results for this antibody can be found at www.proteinatlas.org

  • All lanes : Anti-HIF-2-alpha antibody (ab20654) at 1 µg/ml

    Lane 1 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
    Lane 2 : PC12 nuclear extract lysate (hypoxia treated) (ab14886)

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 96 kDa
    Observed band size: 96 kDa
    Additional bands at: 240 kDa, 25 kDa, 45 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 20 minutes

References

This product has been referenced in:
  • Wu L  et al. Overexpression and correlation of HIF-2a, VEGFA and EphA2 in residual hepatocellular carcinoma following high-intensity focused ultrasound treatment: Implications for tumor recurrence and progression. Exp Ther Med 13:3529-3534 (2017). Read more (PubMed: 28587437) »
  • Prieto-Domínguez N  et al. Melatonin enhances sorafenib actions in human hepatocarcinoma cells by inhibiting mTORC1/p70S6K/HIF-1a and hypoxia-mediated mitophagy. Oncotarget 8:91402-91414 (2017). Read more (PubMed: 29207653) »
See all 13 Publications for this product

Customer reviews and Q&As

1-10 of 13 Abreviews or Q&A

Question

Dear Kate



Thanks for getting back to me. I was told that this testing had been done in house so this is why this antibody was recommended as you would be able to give me precise instructions. I am disappointed to find this is not the case as we need the results for this antigen urgently for a grant application.



Is there a contact phone number I can call to get a quick response?



Regards



Susan


__________________________________________________



Dr Susan J Wilson PhD

Senior Lecturer

Head of the Histochemistry Research Unit

andBMedSc Coordinator



Contact Details: Histochemistry Research Unit,

Sir Henry Wellcome Laboratories,

Mailpoint 894, Level B, South Block,

Southampton General Hospital

Tremona Road

Southampton

SO16 6YD



Tel: 023 8079 6316

Fax: 023 8079 5016

http://www.hru.soton.ac.uk

P Think Green - Do you really need to print this e-mail?






From: technical@abcam.com [mailto:technical@abcam.com]
Sent: 21 August 2012 14:48
To: Wilson S.J.
Subject: Reply from Abcam to your enquiry regarding ab20654, ab109731 [CCE4072756]



















https://www.abcam.com/index.html?utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header









Dear Susan,

Thank you for your message.

I am sorry the testing was performed by Protein Atlas in this particular case, as stated on the datasheet. We therefore regrettablyhave no further information on this occasion. I am aware however that they do the antibody testing usingautomated staining machines and set protocols.I can suggest perhaps you may like to contact them for further information:

mailto:contact@proteinatlas.org

With regards to the length of antigen retrieval, this is something that can sometimes require optimization. Using shorter time of 2, 5, or 10 minutes can sometimes provide better results.

I am sorry I have no more details to send on this occasion.If you have any further questions, please do not hesitate to contact me.


Help us improve our service.
https://www.abcam.com/index.html?pageConfig=technicalSurvey&intCCEID=4072756


Best regards,
Kate

Kate Hayes
Scientific Support Supervisor
Abcam plc
https://www.abcam.com


Your original inquiry to Abcam:


Dear Kate


Thanks for the quick reply.

Do you have any further details about the antigen retrieval? Was the heat by microwave and for how long? Guilliame thought we may be over cooking ours by using 20 minutes.

Regards

Susan




__________________________________________________



Dr Susan J Wilson PhD

Senior Lecturer

Head of the Histochemistry Research Unit

andBMedSc Coordinator



Contact Details: Histochemistry Research Unit,

Sir Henry Wellcome Laboratories,

Mailpoint 894, Level B, South Block,

Southampton General Hospital

Tremona Road

Southampton

SO16 6YD



Tel: 023 8079 6316

Fax: 023 8079 5016

http://www.hru.soton.ac.uk

P Think Green - Do you really need to print this e-mail?






From: mailto:technical@abcam.com mailto:[mailto:technical@abcam.com]
Sent: 21 August 2012 08:51
To: Wilson S.J.
Subject: Reply from Abcam to your enquiry regarding ab20654, ab109731 [CCE4071608]



















https://www.abcam.com/index.html?utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header









Dear Dr Wilson

Thank you for your telephone call yesterday and for your patience. I am sorry no one had got back to you sooner.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested on the telephone, I have issued a free of charge replacement of alternative ab20654 with the order number 1149392.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our 6 month Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

With regards to the antigen retrieval method used for IHC-P with ab20654, I can confirm this application has been added from data and results for his antibodyfrom the Protein Atlas website, which isshown onour datasheet. This includes the following information:

'Antigen retrieval was performed by heat induction in citrate buffer pH 6'

I hope this will be helpful. Please let me know if you have any further questions and I wish you the best of luck with yourexperiments.




Help us improve our service.
https://www.abcam.com/index.html?pageConfig=technicalSurvey&intCCEID=4071608


Best regards,
Kate

Kate Hayes
Scientific Support Supervisor
Abcam plc
https://www.abcam.com


Your original inquiry to Abcam:


ab109731 Anti-HIF2 alpha antibody

- no staining at all
- human tissue sections
- Tried microwave for 20 minutes
- Do not have solution left to try other troubleshooting steps




Abcam Customer Services and Scientific Support Team
https://www.abcam.com/index.html?pageconfig=technical&utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Body

[CCE4071608]














Discover more at abcam.com





























Abcam Customer Services and Scientific Support Team
https://www.abcam.com/index.html?pageconfig=technical&utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Body

[CCE4072756]













Discover more at abcam.com

Read More
Answer

Thank you for your reply.

I apologise for any confusion, I have confirmed again with our laboratories that this has not been tested in IHC-P in house. I understand this information is important for you and I am sincerely sorry we are not able to provide further details on this occasion.

As discussed in the previous email, I can suggest perhaps you may like to consider contacting Protein Atlas for further information regarding the testing method they used. They can be contacted on the following email address. I'm sorry I am not able to find a phone contact on their website.

mailto:contact@proteinatlas.org

Please do not hesitate to let me know if there is anything further I can help with.

Read More

Answer

Thank you for your message.

I am sorry the testing was performed by Protein Atlas in this particular case, as stated on the datasheet. We therefore regrettablyhave no further information on this occasion. I am aware however that they do the antibody testing usingautomated staining machines and set protocols.I can suggest perhaps you may like to contact them for further information:

mailto:contact@proteinatlas.org

With regards to the length of antigen retrieval, this is something that can sometimes require optimization. Using shorter time of 2, 5, or 10 minutes can sometimes provide better results.

I am sorry I have no more details to send on this occasion.If you have any further questions, please do not hesitate to contact me.

Read More

Answer

Thank you for your telephone call yesterday and for your patience. I am sorry no one had got back to you sooner.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested on the telephone, I have issued a free of charge replacement of alternative ab20654 with the order number 1149392.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our 6 month Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

With regards to the antigen retrieval method used for IHC-P with ab20654, I can confirm this application has been added from data and results for his antibodyfrom the Protein Atlas website, which isshown onour datasheet. This includes the following information:

'Antigen retrieval was performed by heat induction in citrate buffer pH 6'

I hope this will be helpful. Please let me know if you have any further questions and I wish you the best of luck with your experiments.

Read More

Question
Answer

Thank you for calling Abcam earlier today.

I am sorry about the issues you are having with ab20654 in IHC-P. If the protocol variations that we talked about did not prove to be effective, then please let me know. The antibody is covered under our Abpromise for six months and is guaranteed to work in IHC-P on human samples . If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

If there is anything else I can help you with, please let me know.

Read More

Answer

Thank you for getting back to me and sorry for the delay in my response.

Could you provide me with the document which refers to the power level 7 which you refer to? I have had some difficulty in finding a reference to this.

Unfortunately we have not performed IHC staining of paraffin embedded sections in house and I am therefore unable to obtain precise details of how the image presented on the datasheet was obtained however, I would suggest initially trying the citrate buffer (pH 6.0) with heating the buffer toboilingwith the slides for3, 5, 10 and20 minutes to see which provides the best staining.

Using a domestic (850W) or scientific microwave, place the slides in the microwave vessel and if using a domestic microwave (850W), put on maximum and wait until the solution comes to the boil. Continue to boil for the recommended time periods above then remove the vessel and run cold water into it. Continue with the staining protocol as usual.

I hope this information has been of help. If you require any further information, please do not hesitate to contact us again.

Read More

Answer

Thank you for contacting us and reporting the problems you have been experiencing in using anti-HIF2 alpha antibody (ab20654) in IHC staining.

The image that you have referred to was staining of human adrenal gland which has the nucleus counter stained in blue and the HIF-2 alpha detected by HRP secondary antibody, in brown. The staining is mainly cytoplasmic. The tissue was treated with heat mediated antigen retrieval using citrate buffer (pH 6)

More staining obtained with ab20654 can be observed from the following site:

http://www.proteinatlas.org/ENSG00000116016

I am sorry to hear that the staining you have observed has not been good. If you would like, I can have a look at the protocol you have been using and the results seen to see if I can make any suggestions to improve the results. If you would like me to do this please fill out the questionnaire I have attached to this email.

If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.

Read More

Answer

Thank you for contacting us. I hope you had a nice Easter weekend!

Firstly, to your question about the quantity of a drop of DABchromogen.To be honest, I am not quite sure what you are referring to with this question. Usually, a drop is the volume that can be transferred with for example a pasteur pipet (something around 50-200 µl, depending on the dropper). Also, afew dropsof liquid might berequired to cover the sections, so the volume depends on the size ofyour tissue sections. If you refer to the protocol of ab80436, I would recommend to follow the protocol which indicates to add 20 µl of the DAB Plus Chromogen to 1 ml of DAB Plus Substrate to prepare the detection solution (step 9).

I am sorry to hear you have been experiencing problems with our HIF2 Alpha antibody. The quality of our products is important to us and I would like to reassure you that we investigate all customer complaints.

Indeed, the staining should be nuclear (as you can for example see at The Protein Atlas, http://www.proteinatlas.org),thus I would like to investigate this particular case further for you, and also obtain more information for our quality records. In order to proceed with this, I have enclosed a questionnaire. I would appreciate if you could complete this. It will help you put the information we require together very easily. I would appreciate if you could also provide an image which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.

Read More

Answer

We usually recommend overnight incubation as thisallows antibodies of lower titer or affinity to be used by simply allowing more time for the antibodies to bind. Also, regardless of the antibody’s titer or affinity for its target, once the tissue has reached saturation point no more binding can take place. Overnight incubation ensures that this occurs.

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

We are always happy to help :-) Have a great day!

Read More

Answer

Thank you for contacting us.

Regarding your questions, I am happy to let you know the following:

1) Positive control for ab20654: HIF2alpha is expressed in most tissues, with highest levels in placenta, lung and heart. Also, as shown on the datasheet it works with adrenal gland tissue. Further details can be found at http://www.proteinatlas.org/ENSG00000116016/normal. Once you have decided on one of these tissues, you can browse our control slides which we have available at https://www.abcam.com/index.html?c=2300. There is for example adrenal normal tissue (ab4282) or adrenal tumour tissue (ab4631) available.
Negative control for these antibodies: For these targets it is almost impossible to find a tissue that does not express these proteins, therefore, a negative control seems not applicable here. However, a negative control which can always be used is the no-primary-control, i.e. a staining with the secondary antibody only to show there is no unspecific binding.

2) We would recommend to aliquot antibodies in a minimum volume of 10 μl in small suitable vials (less is not advisable due to loss caused by adherence to the tube wall and/or evaporation). For further details see our Antibody storage guide (attached).

3) Aliquots or dilutions for storage of the kit ab80436 are not required. Only the concentration of the primary antibody needs to be optimised, preferably by a dilution series.

4) The optimal antigen retrieval conditions depend on many factors and can vary from antibody to antibody, from tissue to tissue etc. This includes the methods and buffers (citrate, tris/EDTA) which result in the optimal antigen retrieval. Therefore, I have attached our IHC protocol for more details (which can also be found online: https://www.abcam.com/index.html?pageconfig=resource&rid=11384)

5) Citrate buffer can be stored at room temperature for 3 months or at 4°C for longer storage.

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

Read More

1-10 of 13 Abreviews or Q&A

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