Overview

  • Product name
  • Description
    Rabbit polyclonal to ATF6
  • Host species
    Rabbit
  • Specificity
    Based on 100% sequence alignment, this antibody detects ATF6 isoform A (alpha). The antibody is predicted to recognize the 50kDa fragment as the it recognizes a peptide sequence from near the amino terminus. We have not tested stressed cells to see if the 50kDa cleavage product can be detected.
  • Tested applications
    Suitable for: ICC, ELISA, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide corresponding to Human ATF6 (N terminal). 17 a.a. immunogen somewhere between a.a. 25-75

  • Positive control
    • MDA MB 361 cell lysate.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer
    Preservative: 0.02% Sodium azide
    Constituent: 99% PBS
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    Immunoaffinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab37149 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
ICC Use a concentration of 10 µg/ml.
ELISA Use at an assay dependent concentration.

Only Peptide ELISA has been tested.

ICC/IF Use a concentration of 20 µg/ml.
WB Use a concentration of 0.5 - 1 µg/ml. Detects a band of approximately 85 kDa (predicted molecular weight: 75 kDa).

Target

  • Function
    Transcription factor that acts during endoplasmic reticulum stress by activating unfolded protein response target genes. Binds DNA on the 5'-CCAC[GA]-3'half of the ER stress response element (ERSE) (5'-CCAAT-N(9)-CCAC[GA]-3') and of ERSE II (5'-ATTGG-N-CCACG-3'). Binding to ERSE requires binding of NF-Y to ERSE. Could also be involved in activation of transcription by the serum response factor.
  • Tissue specificity
    Ubiquitous.
  • Sequence similarities
    Belongs to the bZIP family. ATF subfamily.
    Contains 1 bZIP domain.
  • Domain
    The basic domain functions as a nuclear localization signal.
    The basic leucine-zipper domain is sufficient for association with the NF-Y trimer and binding to ERSE.
  • Post-translational
    modifications
    During unfolded protein response an approximative 50 kDa fragment containing the cytoplasmic transcription factor domain is released by proteolysis. The cleavage seems to be performed sequentially by site-1 and site-2 proteases.
    N-glycosylated. The glycosylation status may serve as a sensor for ER homeostasis, resulting in ATF6 activation to trigger the unfolded protein response (UPR).
    Phosphorylated in vitro by MAPK14/P38MAPK.
  • Cellular localization
    Endoplasmic reticulum membrane and Nucleus. Under ER stress the cleaved N-terminal cytoplasmic domain translocates into the nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • Activating transcription factor 6 alpha antibody
    • Activating transcription factor 6 antibody
    • ATF 6 antibody
    • ATF6 alpha antibody
    • ATF6 antibody
    • ATF6-alpha antibody
    • ATF6A antibody
    • ATF6A_HUMAN antibody
    • cAMP dependent transcription factor ATF 6 alpha antibody
    • cAMP-dependent transcription factor ATF-6 alpha antibody
    • Cyclic AMP dependent transcription factor ATF 6 alpha antibody
    • DKFZp686P2194 antibody
    • ESTM49 antibody
    • FLJ21663 antibody
    • Processed cyclic AMP dependent transcription factor ATF 6 alpha antibody
    • Processed cyclic AMP-dependent transcription factor ATF-6 alpha antibody
    see all

Images

  • All lanes : Anti-ATF6 antibody (ab37149) at 0.5 µg

    Lane 1 : Untreated HeLa Cell Lysate
    Lane 2 : Tunicamycin-treated-2mg HeLa Cell Lysate
    Lane 3 : Tunicamycin-treated-20mg HeLa Cell Lysate

    Predicted band size: 75 kDa

  • ab37149 staining human Hella cells by ICC/IF. Cells were PFA fixed and permeabilized for 10 minutes in 0.1% Triton X-100 prior to blocking in 3% BSA for 15 minutes at 25°C. The primary antibody was diluted 1/200 and incubated with the sample for 1 hour. The secondary antibody used was Alexa Fluor®568 goat anti-rabbit (ab175471) diluted at 1/1000.

    See Abreview

  • A20 (mouse reticulum sarcoma cell line) cells stained for ATF6 using ab37149 at 10 µg/ml in ICC.

  • HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained for ATF6 (green) using ab37149 at 1/100 dilution in ICC/IF, followed by Goat anti-rabbit Alexa Fluor® 488.

    See Abreview

References

This product has been referenced in:
  • Li PC  et al. Seawater inhalation induces acute lung injury via ROS generation and the endoplasmic reticulum stress pathway. Int J Mol Med 41:2505-2516 (2018). Read more (PubMed: 29436612) »
  • Wang B  et al. The Neuroprotection of Low-Dose Morphine in Cellular and Animal Models of Parkinson's Disease Through Ameliorating Endoplasmic Reticulum (ER) Stress and Activating Autophagy. Front Mol Neurosci 11:120 (2018). Read more (PubMed: 29731707) »
See all 38 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Answer

Thank you very much for your email.

We are very sorry to hear that you have problems with our antibodies. I will continue to investigate this issue with you. Please be assured, should we find that any of these antibodies would not work as expected, we will take the necessary actions. We do also guarantee all our products, and will replace the faulty product to you.

I have not found any indication that we would have sent a wrong antibody to you. The epitope of the monoclonal antibody ab11909is not known. That antibody was made against a full-length recombinant ATF6 protein and theepitope has not been mapped to our knowledge. We are also not aware of any specificity issues with this antibody, it has been tested against 3T3 cells in Western blot. We know that this antibody recognizes both full length and active/Cleaved ATF6.

The polyclonal antibody ab37149 has been raised against an immunogen derived from N terminus. It detects the alpha isoform.Due to the immunogen location it isalso predicted to to recognize the 50kD fragment.

I am not sure what the exact expected localisation of ATF6 should be? I have found this publication, however I am not sure how this relates to your results: "In 3-month-old lenses, ATF6 expression is still detectable in lens epithelium and fiber cells hthttp://www.sciencedirect.com/science/article/pii/S1567133X10001110"

Which antibody do you think provides you with the more specific staining? I can also offer to help to troubleshoot the protocol. Have you made the isotype controls also for each of the antibody - would there be anything technical we could improve? I would be pleased to look through the protocols as well. If you wish so, please do fill the attached questionnaire and send it back with an image of the results. This will then allow me to investigate this issue.

I look forward to hear back from you with your thoughts on this. Thank you for your cooperation.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (retina)
Permeabilization
Yes - 0.1% Triton X-100/0.1% sodium citrate
Specification
retina
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 22°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Dec 28 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (retina)
Gel Running Conditions
Non-reduced Denaturing
Loading amount
10 µg
Treatment
TGX gel
Specification
retina
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Dec 28 2016

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 with one unit of ab65838,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.

Read More

Answer

Thank you very much for your follow up email. I am sorry, that it took me so long to look into this.

Can you please comment on the following points:

1.) The expected Golgi and ER staining of the ab11909 could maybe not be observed in not very actively cycling cells. I would expect, that this signal would however be detected in ER and Golgi in cancer cells, such as the MCF7 cells. You might could do a positive control on such a cancer cell line.

2.) In regards to the ab37149, maybe there was a problem with this antibody vial. Although we do not have any indications that there would have been any problems, I can offer you to send you an alternative antibody for free. (and we would appreciate ifyoucould then provide us also with feedbacks on these via abreviews, https://www.abcam.com/abreviews) I could send you a free unit of ab65838, which is also against the N terminal and which therefore should provide a similar staining. The ab65838 has not been tested on frozen sections however. The ab65838 has been tested in Western blot and paraffin embedded tissue sections. It is very possible to work on frozen sections, however this needs to be tested experimentally first.

Please let me know what you think about this offer. I look forward hearing back from you.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry
Sample
Human Cultured Cells (HELA)
Specification
HELA
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% TRITON-x
Blocking step
BSA as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Jan 21 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - other (HELA)
Loading amount
20 µg
Specification
HELA
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Jan 19 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton X-100 (10 mins)
Blocking step
BSA as blocking agent for 15 minute(s) · Concentration: 3% · Temperature: RT°C

Dr. Dan Tattersall

Verified customer

Submitted Nov 14 2008

Answer

Thank you for your reply. Unfortunately we do not know of a suitable positive control for the 50kDa band and have yet to test the antibody for the cleavage fragment. I hope that the recommendations I sent to you regarding protocol changes will help you detect the 50kDa band (use of a RIPA buffer, boil samples in loading buffer at 95-100C for 5 minutes, changing the blocking step, incubating the primary antibody overnight at 4C more concentrated, use Ecl+ detection kit and check the secondary antibody). Good luck with your experiments,

Read More

Answer

Thank you for your enquiry. I am sorry to hear you are having a problem with ab37149. Unfortunately the attachment you have mentioned in your form did not reach us, could you please send it to my attention so I can better understand your problem? I would like to already suggest the following modifications to your protocol: -run a positive control, as it may be that currently your lysate does not contain high levels of the transcription factor. The antibody was originally tested on MDA MB 361 cell lysate so this is a recommended positive control. -As the protein is located in the ER and in the nucleus, I suggest you switch lysis buffer to a RIPA buffer, as this is stronger and will extract the protein very effectively, increasing its levels in your lysate. -We recommend to boil samples in loading buffer at 95-100C for 5 minutes. It may be that currently the heating to 70C is not sufficient to effectively denature the protein, hence it is not recognized well by the antibody. -Try blocking with 5% BSA as some antibodies bind to the milk and hence do not bind to the membrane. -Incubate the primary antibody overnight at 4C. It would be worth trying more concentrated antibody, for example dilutions of 1:500, 1:1000. -We recommend to use Ecl+ or more sensitive kits such as the super signal Pierce kits, as we find that ECl is not very sensitive. Please let me know if this helps and do not hesitate to contact us for further advice. -Check the secondary antibody works with other primary antibodies, as it may be responsible for the low signal if its HRP activity is diminished. I hope these recommendations will already help you, please do not hesitate to contact me if I can be of further assistance.

Read More

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