Anti-HIF-2-alpha抗体(ab199)

Reviews (6)Q&A (15)References (85)

Key features and details

  • Rabbit polyclonal to HIF-2-alpha
  • Suitable for: ICC/IF, WB
  • Reacts with: Mouse, Rat, Human
  • Isotype: IgG

概述

  • 产品名称

    Anti-HIF-2-alpha抗体
    参阅全部 HIF-2-alpha 一抗

  • 描述

    兔多克隆抗体to HIF-2-alpha

  • 宿主

    Rabbit

  • 特异性

    Specific for HIF-2-alpha / EPAS 1. Does not cross react with HIF-1-alpha.

  • 经测试应用

    适用于: ICC/IF, WBmore details

  • 种属反应性

    与反应: Mouse, Rat, Human

  • 免疫原

    Synthetic peptide corresponding to Mouse HIF-2-alpha aa 632-646.
    Sequence:

    GRSNTQWPPDPPLHF

    Run BLAST with BLAST the sequence with ExPASy Run BLAST with BLAST the sequence with NCBI

性能

应用

Our Abpromise guarantee covers the use of ab199 in the following tested applications.

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

应用 Ab评论 说明
ICC/IF Use a concentration of 1 µg/ml.
EMSA Use at an assay dependent concentration.
WB 1/200 - 1/1000. Detects a band of approximately 118 kDa (predicted molecular weight: 100 kDa). The following should be taken into consideration: sufficient hypoxia induction is necessary; use nuclear extracts for cleaner results; use positive/negative controls to see which band is up-regulated; HIF2a degrades rapidly (fast sample preparation, protease inhibitors!).

靶标

  • 功能

    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.

  • 组织特异性

    Expressed in most tissues, with highest levels in placenta, lung and heart. Selectively expressed in endothelial cells.

  • 疾病相关

    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.

  • 序列相似性

    Contains 1 basic helix-loop-helix (bHLH) domain.
    Contains 1 PAC (PAS-associated C-terminal) domain.
    Contains 2 PAS (PER-ARNT-SIM) domains.

  • 翻译后修饰

    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.

  • 细胞定位

    Nucleus.
  • Information by UniProt

  • 数据库链接

    see all

  • 别名

    • 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

图片

  • Western blot - Anti-HIF-2-alpha antibody (ab199)
    Western blot - Anti-HIF-2-alpha antibody (ab199)

    Analysis on normoxic and hypoxic nuclear rat cell lysates.

  • Western blot - Anti-HIF-2-alpha antibody (ab199)
    Western blot - Anti-HIF-2-alpha antibody (ab199)Image from PLoS One. 2012; 7(1): e29876. Fig3A, doi: 10.1371/journal.pone.0029876 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    HIF-2α levels in HIF-2α−/− and WT mouse kidneys.

    HIF-2α−/− mice or their wild-type littermates were exposed to left ureteral obstruction (UO), which continued for 24 hours, and then was released. 2 days after release of UO or at the corresponding time point in the non-UO sham-operated mice, the left kidneys were harvested and subjected to immunoblot analyses of HIF-2α and co-detection of TBP as a loading control

    Nuclear extracts were isolated from harvested whole kidneys using NE-PER Nuclear and Cytoplasmic Extraction Reagents supplemented with Complete Protease Inhibitor Cocktail Tablets. Nuclear protein fractions were electrophoresed on 10% SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. Membranes were blocked with LI-COR blocking buffer (LI-COR Biosciences, Lincoln, NE). Membranes were then incubated with the same blocking solution containing rabbit polyclonal primary antibodies against HIF-2α (1:500, ab199). After washing, membranes were incubated at room temperature for 1 h in TBS/0.05% Tween 20 buffer with the IRDye800 secondary antibodies (1:10000) and then washed again in TBS/0.05% Tween 20 for 3 times. The blot was visualized using an Odyssey infrared imaging system. All values were normalized to a loading control TATA binding protein (TBP, 1:2000, ab818, Abcam) and expressed as fold increase relative to control.

  • Immunocytochemistry/ Immunofluorescence - Anti-HIF-2-alpha antibody (ab199)
    Immunocytochemistry/ Immunofluorescence - Anti-HIF-2-alpha antibody (ab199)

    ICC/IF image of ab199 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab199, 1µg/ml) overnight at +4°C. 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) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Western blot - Anti-HIF-2-alpha antibody (ab199)
    Western blot - Anti-HIF-2-alpha antibody (ab199)This image is courtesy of Marilo Dolores Chiara
    All lanes : Anti-HIF-2-alpha antibody (ab199) at 1/500 dilution

    Lane 1 : 20ug of whole cell lysate from PC12 cells.
    Lane 2 : 20ug of whole cell lysate from PC12 cells exposed to hypoxic conditions (12 hours at 3% oxygen).
    Lane 3 : 20ug of whole cell lysate from PC12 cells exposed to hypoxic conditions (12 hours at 3% oxygen) and incubated with antisense HIF2 alpha oligonucleotides 72 hours prior to treatment.

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 100 kDa
    Observed band size: 96 kDa
    why is the actual band size different from the predicted?



    The blots were probed with anti-alpha tubulin as a loading control.

    See Abreview

文献 (85)

发表研究结果有使用 ab199？请让我们知道，以便我们可以引用本数据表中的参考文章。

ab199 被引用在 85 文献中.

  • Du X  et al. Hypoxia-Inducible Factor 1a and 2a Have Beneficial Effects in Remote Ischemic Preconditioning Against Stroke by Modulating Inflammatory Responses in Aged Rats. Front Aging Neurosci 12:54 (2020). PubMed: 32210788
  • Luo Y  et al. CD146-HIF-1a hypoxic reprogramming drives vascular remodeling and pulmonary arterial hypertension. Nat Commun 10:3551 (2019). PubMed: 31391533
  • Wang Y  et al. Berberine improves insulin resistance in adipocyte models by regulating the methylation of hypoxia-inducible factor-3a. Biosci Rep 39:N/A (2019). PubMed: 31652442
  • Kung-Chun Chiu D  et al. Hypoxia regulates the mitochondrial activity of hepatocellular carcinoma cells through HIF/HEY1/PINK1 pathway. Cell Death Dis 10:934 (2019). PubMed: 31819034
  • Chowdhury D  et al. Metallothionein 3 Controls the Phenotype and Metabolic Programming of Alternatively Activated Macrophages. Cell Rep 27:3873-3886.e7 (2019). PubMed: 31242420
View all Publications for this product

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1-10 of 21 Abreviews or Q&A

Western blot abreview for Anti-HIF-2-alpha antibody - ChIP Grade

 Average
Abreviews
abreview image
Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing (10% SDS PAGE)
Sample
Mouse Tissue lysate - whole (Mouse brain)
Specification
Mouse brain
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Abcam user community

Verified customer

提交于 Jun 02 2014

Western blot abreview for Anti-HIF2 alpha antibody - ChIP Grade

 Inconclusive
Abreviews
Application
Western blot
Sample
Human Tissue lysate - whole (Heart, Left Ventricle)
Loading amount
40 µg
Specification
Heart, Left Ventricle
Gel Running Conditions
Reduced Denaturing (4-12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Dawn Bowles

Verified customer

提交于 Jan 10 2013

ChIP abreview for Anti-HIF-2-alpha antibody - ChIP Grade

 Excellent
Abreviews
abreview image
Application
ChIP
Sample
Human Cell lysate - nuclear (Kidney (Clear Cell Renal Carcinoma))
Negative control
Parental clear cell renal carcinoma cells (786-O).
Specification
Kidney (Clear Cell Renal Carcinoma)
Detection step
Other
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% Formaldehyde
Positive control
Metastatic clear cell renal carcinoma cells (M1A).
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Paulo Rodrigues

Verified customer

提交于 Aug 17 2018

Question


the customer would like to ask some more clarification:
- Which is the the active form of HIF-2a (the band at 96 KDa or the other band at 120 kDa)?
- Which are the post-translational modifications (phosphorilations acetylation, methylation, glycosylation, sumoylation, ubiquitination, etc.)?

Thanks in advance and kind regards

Read More
Answer

Thank you for your reply.

As discussed, the HIF1 and 2 alpha subunits have been shown to be present in forms between 96 and 120 kDa through postranslational modifications. This has been studied in more depth with HIF1-alpha than HIF2-alpha. For example, in the following study they found a band at ˜90 kDa and one at ˜116 kDa in renal cell carcinoma cell line:

http://clincancerres.aacrjournals.org/content/11/3/1129.full

They found the ratio between the two bands differed depending on the cell lines tested.

This topic can be reviewed in more detail from literature but I believe both forms are active.

I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

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Question

Dear Sirs,
a customer of ours has bought your ab199 and found by WB two bands (one at 95kDa and one at 120kDa).
In your datasheet it is indicated: "For instance, unprocessed HIF2 is 96 KDa while the full post-translationally modified form is 118 KDa or larger".
The customer would like to know what do you mean with "unprocessed HIF2" and moreover which are the full post-translationally modified forms?

Thanks in advance and kind regards

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Answer

Many thanks for contacting us and your interest in our products.

Both the HIF-1alpha and HIF-2alpha have been to show this characteristics. Whilst the expressed protein would be expected at ˜96 kDa, this can be post-translationally modified, increasing the protein size. Bands are therefore commonly observed between 96 kDa to 120 kDa.

The level of post-translational modifications can be as a result of the compounds used to increase HIF production as well as the cell strains probed.

I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

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Question

Thank you for your fast reply. The old PO number was: If you are going to send a replacement product, please send it to this free-of-charge PO number:

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement through Cedarlane of ab109616.

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

I wish you the best of luck with your research.

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Question

Amount: 200ug (Mouse lung tissue, animals in chronic hypoxia for 3 weeks) Blocking conditions: 5% milk in 1x TBST, 1hr, Detection method: secondary donkey anti-rabbit antibody Electrophoresis: 8% gel Transfer: Wet/2hrs. Primary: 1:500, overnight (~20hrs), 4oC. Secondary: 1 hr, room temp, 1:2500 Wash steps: three TBST after primary, three after secondary, one PBS I did not detect any bands at the correct size at all (some band at 60kDa). I did this western at the same time as for HIF1a (product: ab2185) I got ALOT of background (I am optimizing now for HIF1a). However, I dont see how this antibody will work since I got 200ug of protein loaded and 1:500 primary and very high secondary (1:2500)!! I also did the beta-actin staining, and it works fine on the same membrane (without stripping) In your sheet, it mentions that the lung is most abundant in the HIF-2a protein!! If possible, instead of a refund, I would like to get this antibody instead: ab109616. Thank you

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Answer

Thank you for contacting us.

I am very sorry to hear that this product did not work as specified on our datasheet and would like to thank you for the information which you have provided. This product is covered by our Abpromise guarantee, provided that it has been purchased within the last six months. In order to send to you a replacement product I will need your Abcam confirmation number or the PO used to purchase this product from Abcam.

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Question

Lot number: GR37292-1

Inquiry: I had ordered anti-HIF-2 alpha antibody with product code#ab199. The order number is *****.
Unfortunately, the performance was not satisfactory. There are two problems. Firstly, we are not always detecting a band of the correct size and secondly, we always obtain a lot of background noise.
Below, there is a description of the anti-HIF-2 alpha Western Blot procedure that we used, in order to find out if the problem is due to any of the protocol’s stages.
We prepared whole cell lysates. The cell lines used were Huh7 and HepG2 and U87. Cells were treated under 1% hypoxia for 8 and 16 hours.
1. We run the proteins (50μg and 100μg) on SDS 8% polyacrylamide gel.
2. Transfered to nitrocellulose membranes. The transfer was verified by ponceau red staining.
3. Blocked membranes for 1.5 hours with 1x western wash buffer (PBS1x + TWEEN) containing 3% BSA. (We have used the same blocking conditions with other antibodies such as anti-HIF1α, anti- actin, anti- tubulin, anti-P- MAPK, etc, so we are pretty sure that it is working).
4. Washed with 1x western wash (PBS1x + TWEEN) for 30 minutes at RT (3 x 10 minutes).
5. Incubated membranes with the antibody HIF2α (#ab199) 1:500 in 1x western wash 1 % BSA overnight at 4Co.
6. Washed with 1x western wash (PBS1x + TWEEN) for 30 minutes at RT (3 x 10 minutes).
7. Incubated membranes with the second antibody (anti-rabbit 1:1000 from Cell Signaling) in 1x western wash (PBS1x + TWEEN) 5% non fat milk.
8. Washed with 1x western wash (PBS1x + TWEEN) for 30 minutes at RT (3 x 10 minutes).
9. For the exposure we used chemiluminescense. We also attach some western blot films.
Also, I would like to mention that we use the ab199 antibody (different Lot numbers) since 2008 and we haven’t any problem until now. Would you be willing to send us a small amount from a different Lot? Thanks in advance for your time.

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Answer

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.
The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.
Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty lot.
As requested, I have issued a free of charge replacement with a new lot of ab199, the order number is **********.
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 Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.
I wish you the best of luck with your research.

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Question

The ab199 replacement vial that I received recently turned also out not to work. I have attached a word document with all the details.

During the last years, I have bought two vials of this antibody that have been working beautifully and since a lot of my data is based on this antibody, I’m really keen to continue with the same antibody. Do you have any lot in stock that you know is behaving well (for western blot) that I could get this non-working vial replaced with?

I would also like to inform you, that If it is possible to deliver an ab199 of the same quality as I’ve experienced before, my plan is to order in two several vials of the same lot and store in -20 (I know it is not recommended to freeze the antibody but I’m willing to take the risk) for future usage.

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Answer

Thank you for confirming these details and for your cooperation.

The details provided enable us to closely monitor the quality of our products.

I am sorry the replacementdid not perform as stated on the datasheet.

As requested, I have issued a free of charge replacement with a new lot. The order number is 1100174.

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 Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Question

Dear abcam,
The ab199 antibody i ordered 24th of April did not work. I have attached a word file with all details.
Please reply all emails to this email, anders.mutvei@ki.se (I have noticed that our lab group share customer number with Helena Lönnqvist (she also works here at Karolinska)). Also, if you send out a new working batch of the ab199 hif-2 alpha, please include my name on the package.

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Answer

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.

I apologize for the inconvenience and as requested, I have issued a free of charge replacement with a different lot number of ab199. Theorder number is *****.

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 Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

1-10 of 21 Abreviews or Q&A

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