Overview

  • Product name
    Anti-Retinoic Acid Receptor alpha antibody
    See all Retinoic Acid Receptor alpha primary antibodies
  • Description
    Goat polyclonal to Retinoic Acid Receptor alpha
  • Host species
    Goat
  • Specificity
    This antibody is expected to recognize all three reported [human] isoforms (NP_000955.1; NP_001019980.1; NP_001028775.1).
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Chicken, Hamster, Cow, Dog
  • Immunogen

    Synthetic peptide:

    CSPSLSPSSH RSSPATQSP

    , corresponding to C terminal amino acids 441-459 of Retinoic Acid Receptor alpha

  • Positive control
    • WB: Human brain lysate. IHC-P: Human cerebellum tissue sections.

Properties

Applications

Our Abpromise guarantee covers the use of ab28767 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/IF 1/50.

See Abreview.

WB Use a concentration of 0.03 - 0.3 µg/ml. Detects a band of approximately 48, 50 kDa (predicted molecular weight: 51 kDa).Can be blocked with Retinoic Acid Receptor alpha peptide (ab4910).
IHC-P Use a concentration of 4 - 6 µg/ml. Steamed antigen retrieval is recommended using citrate buffer pH 6.0.

Target

  • Function
    Receptor for retinoic acid. Retinoic acid receptors bind as heterodimers to their target response elements in response to their ligands, all-trans or 9-cis retinoic acid, and regulate gene expression in various biological processes. The RXR/RAR heterodimers bind to the retinoic acid response elements (RARE) composed of tandem 5'-AGGTCA-3' sites known as DR1-DR5. In the absence of ligand, the RXR-RAR heterodimers associate with a multiprotein complex containing transcription corepressors that induce histone acetylation, chromatin condensation and transcriptional suppression. On ligand binding, the corepressors dissociate from the receptors and associate with the coactivators leading to transcriptional activation. RARA plays an essential role in the regulation of retinoic acid-induced germ cell development during spermatogenesis. Has a role in the survival of early spermatocytes at the beginning prophase of meiosis. In Sertoli cells, may promote the survival and development of early meiotic prophase spermatocytes. In concert with RARG, required for skeletal growth, matrix homeostasis and growth plate function (By similarity). Regulates expression of target genes in a ligand-dependent manner by recruiting chromatin complexes containing MLL5. Mediates retinoic acid-induced granulopoiesis.
  • Involvement in disease
    Note=Chromosomal aberrations involving RARA are commonly found in acute promyelocytic leukemia. Translocation t(11;17)(q32;q21) with ZBTB16/PLZF; translocation t(15;17)(q21;q21) with PML; translocation t(5;17)(q32;q11) with NPM. The PML-RARA oncoprotein requires both the PML ring structure and coiled-coil domain for both interaction with UBE2I, nuclear microspeckle location and sumoylation. In addition, the coiled-coil domain functions in blocking RA-mediated transactivation and cell differentiation.
  • Sequence similarities
    Belongs to the nuclear hormone receptor family. NR1 subfamily.
    Contains 1 nuclear receptor DNA-binding domain.
  • Domain
    Composed of three domains: a modulating N-terminal domain, a DNA-binding domain and a C-terminal ligand-binding domain.
  • Post-translational
    modifications
    Phosphorylated on serine and threonine residues. Phosphorylation does not change during cell cycle. Phosphorylation on Ser-77 is crucial for transcriptional activity (By similarity). Phosphorylation by AKT1 is required for the repressor activity but has no effect on DNA binding, protein stability nor subcellular localization. Phosporylated by PKA in vitro. This phosphorylation on Ser-219 and Ser-369 is critical for ligand binding, nuclear localization and transcriptional activity in response to FSH signaling.
    Sumoylated by SUMO2, mainly on Lys-399 which is also required for SENP6 binding. On all-trans retinoic acid (ATRA) binding, a confromational change may occur that allows sumoylation on two additional site, Lys-166 and Lys-171. Probably desumoylated by SENP6. Sumoylation levels determine nuclear localization and regulate ATRA-mediated transcriptional activity.
    Trimethylation enhances heterodimerization with RXRA and positively modulates the transcriptional activation.
    Ubiquitinated.
  • Cellular localization
    Nucleus. Cytoplasm. Nuclear localization depends on ligand binding, phosphorylation and sumoylation. Transloaction to the nucleus in the absence of ligand is dependent on activation of PKC and the downstream MAPK phosphorylation.
  • Information by UniProt
  • Database links
  • Alternative names
    • NR1B1 antibody
    • Nuclear mitotic apparatus protein retinoic acid receptor alpha fusion protein antibody
    • Nuclear receptor subfamily 1 group B member 1 antibody
    • Nucleophosmin retinoic acid receptor alpha fusion protein NPM RAR long form antibody
    • RAR alpha antibody
    • RAR antibody
    • RAR-alpha antibody
    • rara antibody
    • RARA_HUMAN antibody
    • RARalpha antibody
    • RARalpha1 antibody
    • Retinoic acid nuclear receptor alpha variant 1 antibody
    • Retinoic acid nuclear receptor alpha variant 2 antibody
    • Retinoic acid receptor alpha antibody
    • Retinoic acid receptor alpha polypeptide antibody
    see all

Images

  • Anti-Retinoic Acid Receptor alpha antibody (ab28767) at 0.03 µg/ml + human brain lysate (30µg total protein per lane)

    Developed using the ECL technique.

    Predicted band size: 51 kDa
    Observed band size: 48,50 kDa
    why is the actual band size different from the predicted?

  • ab28767 at 4µg/ml staining Retinoic Acid Receptor alpha in human cerebellum tissue. Steamed antigen retrieval using citrate buffer pH6 was performed. AP staining. Image shows staining of Purkinje cytoplasm.

References

This product has been referenced in:
  • Bertini Teixeira M  et al. Fasciculation and elongation zeta-1 protein (FEZ1) interacts with the retinoic acid receptor and participates in transcriptional regulation of theHoxb4gene. FEBS Open Bio 8:4-14 (2018). Read more (PubMed: 29321952) »
  • Chen J  et al. Inhibition of cancer stem cell like cells by a synthetic retinoid. Nat Commun 9:1406 (2018). Read more (PubMed: 29643385) »
See all 13 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Sheep Tissue sections (intestine)
Antigen retrieval step
None
Permeabilization
No
Specification
intestine
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jun 02 2017

Answer

Thank you for your response.

Again let me emphasize that I am sorry this product is not performing. These complaints are taken seroiusly and this will be looked into by the appropriate groups. I am sorry that the product did not work against the peptide, which I am sure you meant was a RAR as a GDNF would not work with this porduct.

Please let me know if there is anything that we might do for you. Have a nice weekend.

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Answer

Thank you for contact me with this further information. I agree that the positvie control looks very poor, however I am hopeful now as we can use that control to clean up the signal. I would recommend using a 5% milk blocking solution for at least 1 hour at room temperature, followed by overnight incubation with the primary. Titering the antibody, prehaps a range between 1:1000- 1:10,000 or even 1:15,000 is advisable as well. If we can eliminate some of the non-specific binding in this way, then use of the blocking peptide should show the specific band for these antibodies. I hope this information helps. Please let me know if you have any questions.

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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. I have issued a free of charge MCF7 Positive Control Lysate (ab3871) with the order number xxx. Please let me know the results of your further experiments. 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
Answer

Thank you for providing these additional details and images.

I would like to send you the postive control for these products. Using the positve control will provide valuable data in this case as having two products against the target both not work is very unusual. I would also suggest trying a 5% milk blocking step instead of the BSA when using this and titering the primaries and the secondary antibody with this control.


I would like to send a whole cell lysate as these products primarily localize in the cytoplasm with nuclear localization dependent uponligand binding, phosphorylation and sumoylation, it may well be that there is not enough of the target in a nuclear extract.

If you could be please send me the order number you recieved for ab28767 and ab76074, I can use that information to send you the positive control lysate.


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

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Question

DESCRIPTION OF THE PROBLEM Multiple bands we also used a peptide for RARa (ab4910) and non of the bands disappeared from the blot. SAMPLE Mouse nuclear extracts PRIMARY ANTIBODY Abcam Goat Polyclonal to RARa (ab28767) used at 1:5000 in 5% BSA incubated overnight at 4 degrees C washed 2x's in TBST (15 min each) and 1x in TBS (briefly). Also used abcam Rabbit monoclonal to RARa (ab76074) used at 1:750 in 5% BSA incubated overnight at 4 degrees C washed 2x's in TBST (15 min each) and 1x in TBS (briefly). DETECTION METHOD IR POSITIVE AND NEGATIVE CONTROLS USED none ANTIBODY STORAGE CONDITIONS Antibody is stored at -30 degrees C in aliquots of 10 ul. SAMPLE PREPARATION Compartmental Protein Extraction Kit was used for the nuclear extracts. The final solution contains 1x Protease Inhibitor. 10 ug of sample was mixed with loading buffer containing Beta mercaptoethanol and heated for 10 min. Solution was then added to 12% precast gel. AMOUNT OF PROTEIN LOADED 10 ug TRANSFER AND BLOCKING CONDITIONS Transfered in 1x TG with methanol at 350 milli Amp for 1 hour and 15 min. Blocking in 5% BSA in TBS for at least 1 hour. SECONDARY ANTIBODY IR detection with antibodies used at 1:10000 in 5% BSA for at least 1 hour. Wash afterwards 2x's in TBST (15 min each) and 1 x in TBS (briefly). HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? We have used multiple different antibodies, including the 2 from abcam (ab18767 and ab76074). We also used an equal volume of peptide as primary antibody for a few blots, and 5x's more peptide than the volume of primary antibody used. ADDITIONAL NOTES The image attached is with antibody (ab28767) and the peptide added. The blot with antibody ab76074 looks the exact same. The blots without peptide added have the exact same bands present. I am curious as to why the bands are not disappearing from the blots.

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Answer

Thank you for contacting Abcam.

I would like to thank you for supplying information about your protocol and I am sorry to hear that ab28767 has not been performing for you. I am most concerned that you found no difference in your blots after peptide blocking. I noted that you stated that you used the product at 1:5000 but that you repeated the experiments 5 times. Did you try changing the concentration of the primary antibody, prehaps using 1:1000, 1:500 or 1:100? While increased concentrations may lead to high non-specific banding it seems that knowing whether the product is recognizing anything would be helpful. Could you also re-send the images to me as they were included in the notification that I recieved.

Please let me know if you have any questions or concerns. I look forward to hearing from you in the future.

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Answer

Thank you for contacting us. Your credit note ID are: Order No: 896459 - Credit note: 188XX Order No: 944015 - Credit note: 188XX Order No: 955405 - Credit note: 188XX I am sorry that this antibody did not perform as stated on the datasheet. I have asked our accounting department to issue a credit note for you, which can be redeemed against the invoice of a future order by passing it on to your purchasing department. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. If you have questions on how to use the credit note, please contact our accounting department. Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department. The credit note ID is for your reference only and does not automatically guarantee the credit. I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.              

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Answer

Thank you for your response. As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased. Could you please confirm the Abcam Order number (or your PON) and the date of purchase? Were these two antibodies shipped together in the same package? I look forward to hearing from you soon.  

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Answer

Thank you for getting back to me and resending the WB images. It looks that the major band(s) are around 40 kDa slightly lower than the expected 50 kDa. These lower bands could either represent modified (processed Retinoic Acid Receptor alpha) or some degradation products.   1) Sample preparation: I understand from your previous e-mail that the tissue lysates were prepared from mouse “extraocular muscle” and  “tibialis anterior”. I assume since RIPA was used as lysis buffer which contained a mixture of proteinse inhibitors so the unwanted proteinases were properly inhibited. The question is if the samples were taken out of the animals quickly enough and stored at low temperature before homogenization/lysate preparation?   2) Splice variants: The other thing to consider is that According to Swiss-Prot;   2 isoforms have been identified and it may well be that the mature protein is processed differently in different tissues. You may wish to take a look at these site for further information: http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.654583 http://www.uniprot.org/uniprot/P11416   3) Positive control: MCF-7 cell or human brain tissue lysate can be used as positive control as the datasheets of these two antibodies indicate. I would advise you to run a good positive control alongside with the mouse samples to see and check if the experimental conditions and the sample preparations are fine. We normally do not offer positive control free of charge, it is not our procedure. However, if you do not get access to these lysates, I can take your complaint as an exemption and could send you a vial depending on stock availability. All our customer feedback, including complaints are monitored weekly by our in house technical support team. If a product is at fault the technical support team will consider removing the product from our catalogue in order to avoid future customer inconvenience. I have checked our internal record to see if we have any quality-related issues with ab28767 or ab76074. It seems that we have not received any similar complaints so not aware of any quality problem. I hope this helps and if I can assist further, please do not hesitate to contact me.

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Question

It will take approximately 5 minutes to complete the questionnaire. Please fill-in all fields so that we can better assist you. 1) Abcam product code: ab28767 and ab76074 2) Abcam order reference number or product batch number: GR1841-4, GR1935-2 3) Description of the problem: Non-specific binding, absence of correct MW bands 4) Sample preparation: Type of sample (whole cell lysates, fraction, recombinant protein%e2: Whole cell lysates from mouse extraocular muscle and tibialis anterior. Lysis buffer: RIPA Buffer (Sigma) Protease inhibitors: Protease Inhibitor Cocktail Tablets (Roche) Phosphatase inhibitors: NA Reducing agent: beta mercaptoethanol Boiling for ≥5 min? yes Protein loaded ug/lane or cells/lane: 30-34 ug protein/lane, depending on gel Positive control: NIH/3T3 Negative control: NA 5) Percentage of gel: 10% Mini-Protean TGX Precast Gel (BIO RAD) Type of membrane: PVDF and Nitrocellulose Protein transfer verified: Yes, via color standard Blocking agent and concentration: 5% BSA in TBST, 2% BSA in TBST Blocking time: at least 1 hour Blocking temperature: Room Temp 6) Primary antibody (If more than one was used, describe in “additional notes%e2��) : Concentration or dilution: 1/750 (monoclonal), 1/5000 (polyclonal) “additional notes:” GAPDH antibody also used at concentration 0.1 ug/ml Diluent buffer: 5% BSA in TBST Incubation time: Overnight Incubation temperature: 4°C 7) Secondary antibody: Species: IR= Goat anti Rabbit or Donkey anti Goat (depending on monoclonal or polyclonal) AP=Anti-goat IgG (1/5000) or Anti-Rabbit IgG (1/10000) Reacts against: Rabbit or Goat Concentration or dilution: 1/1000 for both IR antibodies, 1/5000 AP anti-goat, 1/10000 AP anti-rabbit Diluent buffer: 5% BSA in TBST Incubation time: at least 1 hour Incubation temperature: Room Temp Fluorochrome or enzyme conjugate: IRDye (IR) or Alkaline Phospatase (AP) 8) Washing after primary and secondary antibodies: Buffer: TBST Number of washes: 2-3 9) Detection method: IR/Alkaline Phosphatase 10) How many times have you run this staining? 14 (4 polyclonal and 10 monoclonal) Do you obtain the same results every time? Yes What steps have you altered to try and optimize the use of this antibody? I have tried both the polyclonal and monoclonal antibodies. I have tried both Nitrocellulose and PVDF membranes. I have tried both 2% and 5% BSA blocks. I have tried imaging with both IR and and alkaline phosphatase detection methods. Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem: Images were attached to original email. Please send this questionnaire by e-mail by cutting and pasting the text in your e-mail (no attachments other than images) to technical@abcam.com.

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Answer

Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am reviewing your enquiries but unfortunately without knowing what exactly MW markers represent it is difficult to assess the bands in the actual samples. Would you be so kind either label the images and resend them to me or clarify the size of the bands on the blot. Are they lower or higher than the expected size (50 kDa)? This information would be crucial for me to process your enquiry properly. I look forward to hearing from you and hope to solve this problem as soon as possible.    

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1-10 of 12 Abreviews or Q&A

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