Overview

  • Product name

    Anti-HIF-1 alpha antibody - ChIP Grade
    See all HIF-1 alpha primary antibodies
  • Description

    Rabbit polyclonal to HIF-1 alpha - ChIP Grade
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-P, WB, IP, IHC-Fr, ICC, ICC/IF, ChIPmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Guinea pig, Human, Xenopus laevis, Monkey
    Predicted to work with: Cow
  • Immunogen

    Fusion protein corresponding to Human HIF-1 alpha aa 432-528.

  • Positive control

    • ICC/IF: HeLa cells. IHC-P: Human kidney tissue. ChIP: HeLa cells. WB: Rat hypoxic nuclear lysate. IP: PC-3 cell lysate treated with cobalt chloride.
  • General notes

    HIF-1 alpha can be a difficult target to work with so we have compiled a summary of all the important information you need to know including useful tips. This can be found in the protocols tab or alternatively click here to download the English version and here to download the Mandarin.

    Under normoxic conditions HIF-1 alpha has a short half-life. It is largely undetectable in cells or tissues grown under normoxic conditions. It is stabilized only at O2 concentrations below 5% and upon stabilization under hypoxic conditions HIF-1 translocates to the nucleus. Therefore we recommend western blots using nuclear extracts and running Hypoxia treated samples as positive control (ab180880). Hypoxia can be induced with treatment using certain agents e.g. CoCl2 or DFO, etc. so proper sample preparation is critical.

Properties

Applications

Our Abpromise guarantee covers the use of ab2185 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
IHC-P 1/100.
WB 1/500 - 1/1000.
IP 1/1000.
IHC-Fr 1/10 - 1/2000.
ICC Use at an assay dependent concentration.
ICC/IF 1/10 - 1/2000.
EMSA 1/1 - 1/100.
ChIP Use at an assay dependent concentration.

25 µl / 15 millions cells

Target

  • Function

    Functions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions activates the transcription of over 40 genes, including, erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. Plays an essential role in embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Activation requires recruitment of transcriptional coactivators such as CREBPB and EP300. Activity is enhanced by interaction with both, NCOA1 or NCOA2. Interaction with redox regulatory protein APEX seems to activate CTAD and potentiates activation by NCOA1 and CREBBP.
  • Tissue specificity

    Expressed in most tissues with highest levels in kidney and heart. Overexpressed in the majority of common human cancers and their metastases, due to the presence of intratumoral hypoxia and as a result of mutations in genes encoding oncoproteins and tumor suppressors.
  • 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.
  • Domain

    Contains two independent C-terminal transactivation domains, NTAD and CTAD, which function synergistically. Their transcriptional activity is repressed by an intervening inhibitory domain (ID).
  • Post-translational
    modifications

    In normoxia, is hydroxylated on Pro-402 and Pro-564 in the oxygen-dependent degradation domain (ODD) by EGLN1/PHD1 and EGLN2/PHD2. EGLN3/PHD3 has also been shown to hydroxylate Pro-564. The hydroxylated prolines promote interaction with VHL, initiating rapid ubiquitination and subsequent proteasomal degradation. Deubiquitinated by USP20. Under hypoxia, proline hydroxylation is impaired and ubiquitination is attenuated, resulting in stabilization.
    In normoxia, is hydroxylated on Asn-803 by HIF1AN, thus abrogating interaction with CREBBP and EP300 and preventing transcriptional activation. This hydroxylation is inhibited by the Cu/Zn-chelator, Clioquinol.
    S-nitrosylation of Cys-800 may be responsible for increased recruitment of p300 coactivator necessary for transcriptional activity of HIF-1 complex.
    Requires phosphorylation for DNA-binding.
    Sumoylated; by SUMO1 under hypoxia. Sumoylation is enhanced through interaction with RWDD3. Desumoylation by SENP1 leads to increased HIF1A stability and transriptional activity.
    Ubiquitinated; in normoxia, following hydroxylation and interaction with VHL. Lys-532 appears to be the principal site of ubiquitination. Clioquinol, the Cu/Zn-chelator, inhibits ubiquitination through preventing hydroxylation at Asn-803.
    The iron and 2-oxoglutarate dependent 3-hydroxylation of asparagine is (S) stereospecific within HIF CTAD domains.
  • Cellular localization

    Cytoplasm. Nucleus. Cytoplasmic in normoxia, nuclear translocation in response to hypoxia. Colocalizes with SUMO1 in the nucleus, under hypoxia.
  • Information by UniProt
  • Database links

  • Alternative names

    • ARNT interacting protein antibody
    • ARNT-interacting protein antibody
    • Basic helix loop helix PAS protein MOP1 antibody
    • Basic-helix-loop-helix-PAS protein MOP1 antibody
    • bHLHe78 antibody
    • Class E basic helix-loop-helix protein 78 antibody
    • HIF 1A antibody
    • HIF 1alpha antibody
    • HIF-1-alpha antibody
    • HIF-1alpha antibody
    • HIF-alpha antibody
    • HIF1 A antibody
    • HIF1 Alpha antibody
    • HIF1 antibody
    • HIF1-alpha antibody
    • HIF1A antibody
    • HIF1A_HUMAN antibody
    • hifla antibody
    • Hypoxia inducible factor 1 alpha antibody
    • Hypoxia inducible factor 1 alpha isoform I.3 antibody
    • Hypoxia inducible factor 1 alpha subunit antibody
    • Hypoxia inducible factor 1 alpha subunit basic helix loop helix transcription factor antibody
    • Hypoxia inducible factor 1, alpha subunit (basic helix loop helix transcription factor) antibody
    • Hypoxia inducible factor1alpha antibody
    • Hypoxia-inducible factor 1-alpha antibody
    • Member of PAS protein 1 antibody
    • Member of PAS superfamily 1 antibody
    • Member of the PAS Superfamily 1 antibody
    • MOP 1 antibody
    • MOP1 antibody
    • PAS domain-containing protein 8 antibody
    • PASD 8 antibody
    • PASD8 antibody
    see all

Images

  • ChIP analysis of HIF-1-alpha genomic sequences from HeLa cells cultivated in normoxic (N) or hypoxic (Hx) conditions, using a HIF1-alpha polyclonal antibody (ab2185). For a negative control, IgG was used and the input as a positive control in the subsequent PCR. Primers for known target genes were used HIF1 alpha, A. EPO and B. VEGF.

  • ICC/IF image of ab2185 stained HeLa cells. The cells were 4% formaldehyde 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 (ab2185, 1/1000 dilution) 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.

  • All lanes : Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)

    Lane 1 : Rat nuclear extract lysate - normoxic
    Lane 2 : Rat nuclear extract lysate - hypoxic
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling HIF-1-alpha with ab2185.

  • All lanes : Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)

    Lane 1 : PC-3 cell lysates - untreated
    Lane 2 : PC-3 cell lysates - treated with 1µl cobalt chloride
    Lane 3 : PC-3 cell lysates - treated with 3µl cobalt chloride
    Lane 4 : PC-3 cell lysates - treated with 5µl cobalt chloride

References

This product has been referenced in:

  • Chen X  et al. Copper promotes the migration of bone marrow mesenchymal stem cells via Rnd3-dependent cytoskeleton remodeling. J Cell Physiol 235:221-231 (2020). Read more (PubMed: 31187497) »
  • Liu FY  et al. TLR9 is essential for HMGB1-mediated post-myocardial infarction tissue repair through affecting apoptosis, cardiac healing, and angiogenesis. Cell Death Dis 10:480 (2019). Read more (PubMed: 31209243) »
See all 123 Publications for this product

Customer reviews and Q&As

11-20 of 44 Abreviews or Q&A

Answer

Thank you for sending the image.

I now think that the bands are an indication of no band. If no protein can be bound antibodies often produce a lot of background.

This background is also often generated by the secondary antibody.

I can suggest that your customer tries the recommendations from my last email and these additional ones:

1.) Do a no primary antibody control top check if the secondary is producing the bands.

2.) Please review the special protocols. A nuclear prep will always give better results with HIFa since it will concentrate the protein and also prevents background by excluding cytoplasmic proteins:

https://www.abcam.com/assets/popups/popup_datasheet_protocols_printer_friendly.cfm?pid=239&intAbId=2185

I hope these recommendation will improve the results. Please let me know if your customer does not get better results after the optimisation of the protocol.

Merry Christmas! :-)

Read More

Answer

Thank you and your customer for taking the time to complete our questionnaire and contact us. I am sorry to hear your customer has had difficulty obtaining satisfactory results from this antibody.

The details your customer has kindly provided will enable us to investigate this case for your customer and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab2185.

As I am sure your customer is aware, HIF1 alpha is difficult target protein to work with. The protein is degraded very quickly and it is therefore very important to treat the samples carefully for the experiment.

1.) I can recommend not to mix blocking agents. Milk in general created less background than BSA, whereas BSA increases the signal.

2.) I suggest to dilute the primary antibody further and to incubate it over night at 4C to achieve the best saturation while maintaining the highest specificity.

3.) Please follow this link to find our recommended protocol for this antibody:
https://www.abcam.com/assets/popups/popup_datasheet_protocols_printer_friendly.cfm?pid=236&intAbId=2185

It would be very helpful for me to see an image of the blot to evaluate which bands are HIF1a. And to give more targeted advise

We are happy to offer this technical support. In the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I hope this information is helpful, thank you and your customer for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Read More

Answer

Thank you for your reply.

I am sorry that ab51608 on original order xxxxxx did not work and we will add a new vial of ab2185 to your next order.

Please accept my apologies for the problems with this antibody and do not hesitate to contact us again if you have any questions.


Read More

Answer

Thank you for taking the time send us this information. Your enquiry has been forwarded to me as my colleague ### is currently away from the office. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for WB and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

Reviewing this case, I would like to offer some suggestions which I hope may help explain the results. I would also appreciate if you can confirm some further details:

1. According to the SwissProt/Uniprot protein database page for this protein, Isoform 1 of the HIF 1 alpha protein has a molecular weight of 92 kDa. There is a smaller isoform of the protein which has a molecular weight of 82 kDa:

http://www.uniprot.org/uniprot/Q16665#Q16665

Looking at the molecular weight markers on the images kindly provided, I can suggest this may be the main band you are observing. I would not normally expect the main HIF 1 alpha band to be above the 98 kDa marker.

2. Is the current vial of secondary antibody working well with other primary antibodies?

3. I can recommend to consider trying BSA rather than milk to block. Changing blocking agent can sometimes help to improve results.

I hope this information is helpful, thank you for your cooperation. If you are still unsatisfied with the results, please do not hesitate to contact me again with further details.

Read More

Question
Answer

Thank you for contacting us on Friday.

As discussed over the phone, unfortunately the anti-HIF1 alpha antibody ab2185 which you were hoping to use in ChIP is not likely to be available until the end of January. The only other antibody which we have shown to work in this application is ab1 but you have already tried this one.

I am afraid there are no other antibodies against this target which we have shown to work in ChIP. This does not mean that they would not work, simply that we have not tired it as yet and would therefore not be able to guarantee its performance. There are a few antibodies which may be worth trying such as the mouse monoclonal ab16066 which has been used in Western blotting, immunoprecipitation and EMSA. Or the rabbit monoclonal ab51608.

If you would share a few more details of the problems you experienced in using ab1 in Western blotting I may be able to provide you with one of these to try free of charge. To this end, could you fill out the questionnaire I have attached to this email? This information will allow us to investigate this case internally and initiate any additional testing where necessary.

I look forward to receiving your reply.

Read More

Answer

Thank you for contacting us.

Unfortunately, because we carry over 90,000 products, it isn't feasible for us to keep small sample sizes of our products, and therefore I am not able to offer a test sample of any of the antibodies requested.

However, the three of them have been tested in immunocytochemistry and immunohistochemistry with mouse samples. Therefore, they are all covered by the Abpromise guarantee.

The Abpromise® guarantee ensures that you can trust our products, and they should work in the tested species and applications stated on the datasheet, or we will offer a replacement, credit, or refund, if reported within 6 months of purchase.

To read about our Abpromise policy: https://www.abcam.com/index.html?pageconfig=abpromise

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

Answer

Thank you for your reply.

As I am sure you are aware, HIF1 alpha is quite a tricky protein to work with. I could not find any Abreviews for the antibody ab2185 used in Western blotting. Where did you come across this information?

In order to see if there is anything I can suggest to help improve the results could you provide me with the following information:

1. Lysis buffer composition:

2. Protease inhibitor used:

3. What oxygen % were the cells cultured under? Which lanes of the blot shared correspond with which time points (left to right 0 hr to 24 hr?).

4. Was a loading control used?

5. How was the primary antibody incubated with the blot (dilution and diluent composition, time and temperature of incubation)?

6. Which secondary antibody was used (dilution and diluent composition, time and temperature of incubation)?

7. Order number (or if yu do not have access to this, the approximate delivery date and the delivery address used)?

This information will also allow us to investigate this case internally and initiate additional testing where necessary.

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.

HIF1 alpha, according to the Uniprot reference Q61221, would be expected to be seen at ˜94 kDa. However, the protein goes through posttranslational modifications and therefore bands up to 120 kDa have been observed. The protein is degraded very quickly and it is therefore very important to treat the samples carefully for the experiment.

Could you share the image of the results obtained and I might be able to comment on if the expected band size is present?

Could I also ask, how are the samples harvested? Were the cells cultured or were they collected from mouse tissue?

Could you describe in some more detail how these samples prepared?

I hope this information has been of some help. I look forward to receiving your reply.

Read More

Question
Answer

Thank you very much for your call today and for letting us know about the trouble with this antibody.

As we discussed, I'm sending a vial of ab51608 free of charge on the order ***, which should arrive tomorrow.

Please keep me updated about the results using this replacement antibody, and if you have any questions or need anything else, please let me know and I'll be happy to help.

Read More

Answer

Thank you for contacting us. I am sorry that ab2185 is producing high background in WB. If you could please let me know your original order or PO number, I will be happy to issue you a refund as requested. Alternatively, I would be happy to send you a free of charge replacement with an alternative antibody to HIF1 alpha, such as ab51608.

For our quality investigation, could you please let me know how your lung tissue was lysed and what buffer was used? Were the samples kept on ice during lysis and were protease inhibitors used? What were the molecular weights of the bands observed? It would be very helpful if you could please send an image of your results.

I will be happy to help you further once I have this additional information.

Read More

11-20 of 44 Abreviews or Q&A

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