Overview

  • Product name
    Anti-HIF3 alpha / IPAS antibody
    See all HIF3 alpha / IPAS primary antibodies
  • Description
    Rabbit polyclonal to HIF3 alpha / IPAS
  • Host species
    Rabbit
  • Specificity
    We have observed both nuclear and cytoplasmic localisation in IF with this antibody and we believe this could be due to dysregulated expression of HIF3 alpha / IPAS in our cell lines.
  • Tested applications
    Suitable for: WB, ICC/IF, IP, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to Human HIF3 alpha/ IPAS aa 350-450 conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab20077)

  • Positive control
    • This antibody gives a positive signal in human heart tissue lysate.
  • General notes

    Previously labelled as HIF3 alpha. 

Properties

Applications

Our Abpromise guarantee covers the use of ab10134 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 1/500. Detects a band of approximately 60 kDa (predicted molecular weight: 72 kDa).Can be blocked with Human HIF3 alpha / IPAS peptide (ab20077).
ICC/IF Use a concentration of 1 - 5 µg/ml.
IP Use at an assay dependent concentration.
IHC-P 1/100.

Target

  • Function
    Involved in adaptive response to hypoxia. Suppresses hypoxia-inducible expression of HIF1A and EPAS1. Binds to core DNA sequence 5'-TACGTG-3' within the hypoxia response element (HRE) of target gene promoters. The complex HIF3A-ARNT activates the transcription of reporter genes driven by HRE. Isoform 4 has a dominant-negative function of inactivating HIF1A-mediated transcription. Isoform 4 attenuates the binding of HIF1A to hypoxia-responsive elements (HRE), thus inhibiting HRE-driven transcription. Hypoxia induces down-regulation of isoform 4, leading to activation of HIF1A in hypoxia. Conversely, upon restoring normoxia, the expression of isoform 4 increases and thereby secure an inhibition of HIF1A activity. Isoform 4 may be a negative regulator of hypoxia-inducible gene expression in the kidney and may be involved in renal tumorigenesis. Functions as an inhibitor of angiogenesis in the cornea.
  • Tissue specificity
    Expressed in kidney. Expressed abundantly in lung epithelial cells. Expression is regulated in an oxygen-dependent manner.
  • Sequence similarities
    Contains 1 basic helix-loop-helix (bHLH) domain.
    Contains 2 PAS (PER-ARNT-SIM) domains.
  • Post-translational
    modifications
    In normoxia, hydroxylated on Pro-492 in the oxygen-dependent degradation domain (ODD) by PHD. The hydroxylated proline promotes interaction with VHL, initiating rapid ubiquitination and subsequent proteasomal degradation.
  • Cellular localization
    Nucleus. Cytoplasm. In the nuclei of all periportal and perivenous hepatocytes. In the distal perivenous zone, detected in the cytoplasm of the hepatocytes.
  • Information by UniProt
  • Database links
  • Alternative names
    • Basic-helix-loop-helix-PAS protein MOP7 antibody
    • bHLHe17 antibody
    • Class E basic helix-loop-helix protein 17 antibody
    • HIF 3A antibody
    • HIF 3A4 antibody
    • HIF-3-alpha antibody
    • HIF3 alpha antibody
    • HIF3-alpha antibody
    • HIF3-alpha-1 antibody
    • HIF3A antibody
    • HIF3A_HUMAN antibody
    • Hypoxia Inducible Factor 3 alpha antibody
    • Hypoxia inducible factor 3 alpha subunit antibody
    • Hypoxia inducible factor three alpha antibody
    • Hypoxia-inducible factor 3-alpha antibody
    • Inhibitory PAS domain protein antibody
    • IPAS antibody
    • Member of PAS protein 7 antibody
    • MOP7 antibody
    • PAS domain-containing protein 7 antibody
    • PASD7 antibody
    see all

Images

  • ICC/IF image of ab10134 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab10134, 5µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

  • ICC/IF image of ab10134 stained HeLa 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 (ab10134, 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.
  • Anti-HIF3 alpha / IPAS antibody (ab10134) at 2 µg/ml + Human heart tissue lysate - total protein (ab29431) at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 72 kDa
    Observed band size: 60 kDa
    why is the actual band size different from the predicted?


    Exposure time: 8 minutes


    This blot was produced using a 10% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% milk before being incubated with ab10134 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.  The band detected by ab10134 migrates at approximately 60-kDa.  Although the predicted molecular weight of this protein is 72-kDa, the 60-kDa band has been observed in the literature.  The intensity of this band has been shown to increase in cells exposed to hypoxic conditions (PMID:16775626). 

    Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

    Datasheets and documents

    References

    This product has been referenced in:
    • Rao K  et al. Protective effect of zinc preconditioning against renal ischemia reperfusion injury is dose dependent. PLoS One 12:e0180028 (2017). WB ; Human . Read more (PubMed: 28686686) »
    • Fala AM  et al. Unsaturated fatty acids as high-affinity ligands of the C-terminal Per-ARNT-Sim domain from the Hypoxia-inducible factor 3a. Sci Rep 5:12698 (2015). Read more (PubMed: 26237540) »
    See all 8 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (small bowel adenoma)
Specification
small bowel adenoma
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate Buffer
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Jul 31 2007

Answer

Thank you for your enquiry. Do you know the name of the person who does the purchasing from your lab? I will only be able to give you a replacement for the antibody. We can only replace or refund the product that was defective, typically only within 90 days of purchase. I did not realize that your purchase was 11 months ago, but of course at this stage, I will honor my promise to replace the antibody. I look forward to receiving your reply. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - other (in vitro translated HIF-3a1)
Specification
in vitro translated HIF-3a1
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4%

Abcam user community

Verified customer

Submitted Feb 22 2006

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Human Cell lysate - other (IVT HIF-3a1)
Specification
IVT HIF-3a1
Immuno-precipitation step
Protein A

Abcam user community

Verified customer

Submitted Feb 07 2006

Answer

Thank you for your enquiry. I blasted the immunogen sequence and found that the sequence is identical in isoforms a, b, c, and d. I was unable to find the other two splice variants. If you could send me the sequences of the other two isoforms, I can find out if they are homologous. Otherwise, if you know that they are conserved within residues 300-400, the antibody should recognize those as well. I hope this information helps, please do not hesitate to contact us if you need any more advice or information,

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Question

Batch number 81145 2)Purchase order number 5893 3)Antibody storage conditions (temperature/reconstitution etc) aliquots stored at-20C 4)Description of the problem (high background, wrong band size, more bands, no band etc.) wrong band and multiple bands as well 5)Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) cell extract,nuclear extract 6)Sample preparation (Buffer/Protease inhibitors/Heating sample etc.) lysis in single lysis buffer contain contain 50mM trisHcl, 150 mM NaCl, 1% triton and protease inhibitor. Samples were heated before loading at 100 C for 5 min 7)Amount of protein loaded from 20 upto 60 µg 8) Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) 10 % gel under reducing condition 9)Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) semidry transfer for 40 min using 20% transfer buffer then block overnight in 5% milk TBST at 4C 10)Primary Antibody (Diluent/Dilution/Incubation time, Wash step) 1:500 in 5% milk TBST for 2 hours then washed with TBST 3 times 10 min each time 11)Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) goat anti mouse HRP used at 1:2000 in milk for 1 hour (Dako) 12)Detection method (ECL, ECLPlus etc.) ECL plus 13)Positive and negative controls used (please specify) positive 293 cells, Negative was Hela cells 14)How many times have you tried the Western? 5-6 times 15)Have you run a "No Primary" control? I used the rabbit serum to detect the exact band 16)What steps have you altered? I tried different concentration of the primary treid to block one hour RT and incubate the primary over night, incubate the secondary at 37C and dilute it further, Prolong the wash step, wash with milk instead

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Answer

Thank you very much for providing the details of your protocol. I would like to try RIPA buffer which contains the ionic detergent sodium deoxycholate as an active constituent and is particularly used for nuclear membrane disruption for nuclear extracts and add a more powerful detergent than triton (NP40 is recommended). Block the membrane in 5% BSA in TBST for 1hour at RT and add a positive control of Jurkat whole cell extract (as seen in the image on the datasheet). Incubate the membrane in primary antibody overnight in TBST (no BSA or milk) at 4C and the secondary in TBST too. I would also like to confirm that it is important to keep samples on ice at all times until addition of the loading buffer and that protease inhibitors can go off, a mixture of various inhibitors is recommended to be added fresh to the lysis buffer. Wash extensively in TBST between incubations of primary and secondary and after incubation of the secondary antibody. Please let me know if this helps and do not hesitate to contact us for further advice,

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Answer

Thank you for your email and I'm sorry to hear that you are experiencing difficulty with ab10134. Could we get a detailed protocol from you, please? We have some general questions, the answers to which will enable us to investigate this matter as quickly as possible. Also, please include the lot number that you received (it is located on the vial) and the Abcam order number or purchase order number that was used. 1. Please describe the problem (high background, wrong band size, more bands, no band etc). 2. On what material are you testing the antibody in WB? •Species? •Cell extract/ Nuclear extract? •Purified protein? •Recombinant protein? 3. How much protein did you load? •How did you prepare the lysate for the analysis (protease inhibitors etc)? •Did you heat the samples? 4. Primary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? 5. Secondary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? •Do you know whether the problems you are experiencing come from the secondary? 6. What detection method are you using? 7. Background bands •Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control) •Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) •At what size are the bands migrating? Could they be degradation products of your target? •Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 8. Optimization attempts •How many times have you tried the Western? •Do you obtain the same results every time e.g. are background bands always in the same place? •What steps have you altered? 9. Did you apply positive and negative controls along with the samples? Please specify.

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