Overview

  • Product name
    Anti-Androgen Receptor antibody - ChIP Grade
    See all Androgen Receptor primary antibodies
  • Description
    Rabbit polyclonal to Androgen Receptor - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    This antibody detects Androgen Receptor. It does not detect estrogen, progesterone, or glucocorticoid receptors. Immunohistochemical staining of AR in rat brain results in intense nuclear staining.
  • Tested applications
    Suitable for: ICC, IHC-Fr, IP, WB, ICC/IF, ChIP, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Hamster, Human, Pig, Non human primates
  • Immunogen

    Fusion protein corresponding to Human Androgen Receptor aa 331-572.

  • Positive control
    • rat brain

Properties

Applications

Our Abpromise guarantee covers the use of ab3509 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 at an assay dependent concentration.
IHC-Fr 1/10 - 1/100.
IP Use at an assay dependent concentration.
WB 1/500. Detects a band of approximately 110 kDa (predicted molecular weight: 110 kDa).
ICC/IF Use at an assay dependent concentration. PubMed: 22850685
ChIP Use at an assay dependent concentration.
IHC-P 1/10 - 1/100.

Target

  • Function
    Steroid hormone receptors are ligand-activated transcription factors that regulate eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Transcription factor activity is modulated by bound coactivator and corepressor proteins. Transcription activation is down-regulated by NR0B2. Activated, but not phosphorylated, by HIPK3 and ZIPK/DAPK3.
    Isoform 3 and isoform 4 lack the C-terminal ligand-binding domain and may therefore constitutively activate the transcription of a specific set of genes independently of steroid hormones.
  • Tissue specificity
    Isoform 2 is mainly expressed in heart and skeletal muscle (PubMed:15634333). Isoform 3 is expressed by basal and stromal cells of prostate (at protein level) (PubMed:19244107).
  • Involvement in disease
    Androgen insensitivity syndrome
    Spinal and bulbar muscular atrophy X-linked 1
    Defects in AR may play a role in metastatic prostate cancer. The mutated receptor stimulates prostate growth and metastases development despite of androgen ablation. This treatment can reduce primary and metastatic lesions probably by inducing apoptosis of tumor cells when they express the wild-type receptor.
    Androgen insensitivity, partial
  • Sequence similarities
    Belongs to the nuclear hormone receptor family. NR3 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. In the presence of bound steroid the ligand-binding domain interacts with the N-terminal modulating domain, and thereby activates AR transcription factor activity. Agonist binding is required for dimerization and binding to target DNA. The transcription factor activity of the complex formed by ligand-activated AR and DNA is modulated by interactions with coactivator and corepressor proteins. Interaction with RANBP9 is mediated by both the N-terminal domain and the DNA-binding domain. Interaction with EFCAB6/DJBP is mediated by the DNA-binding domain.
  • Post-translational
    modifications
    Sumoylated on Lys-388 (major) and Lys-521. Ubiquitinated. Deubiquitinated by USP26. 'Lys-6' and 'Lys-27'-linked polyubiquitination by RNF6 modulates AR transcriptional activity and specificity.
    Phosphorylated in prostate cancer cells in response to several growth factors including EGF. Phosphorylation is induced by c-Src kinase (CSK). Tyr-535 is one of the major phosphorylation sites and an increase in phosphorylation and Src kinase activity is associated with prostate cancer progression. Phosphorylation by TNK2 enhances the DNA-binding and transcriptional activity and may be responsible for androgen-independent progression of prostate cancer. Phosphorylation at Ser-83 by CDK9 regulates AR promoter selectivity and cell growth. Phosphorylation by PAK6 leads to AR-mediated transcription inhibition.
    Palmitoylated by ZDHHC7 and ZDHHC21. Palmitoylation is required for plasma membrane targeting and for rapid intracellular signaling via ERK and AKT kinases and cAMP generation.
  • Cellular localization
    Nucleus. Cytoplasm. Predominantly cytoplasmic in unligated form but translocates to the nucleus upon ligand-binding. Can also translocate to the nucleus in unligated form in the presence of RACK1.
  • Information by UniProt
  • Database links
  • Form
    There are 2 isoforms produced by alternative splicing. Isoform 1 is also known as: AR-B; isoform 2 is known as AR-A or variant AR45.
  • Alternative names
    • AIS antibody
    • ANDR_HUMAN antibody
    • Androgen nuclear receptor variant 2 antibody
    • Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease) antibody
    • Androgen receptor antibody
    • androgen receptor splice variant 4b antibody
    • AR antibody
    • AR8 antibody
    • DHTR antibody
    • Dihydro testosterone receptor antibody
    • Dihydrotestosterone receptor (DHTR) antibody
    • Dihydrotestosterone receptor antibody
    • HUMARA antibody
    • HYSP1 antibody
    • KD antibody
    • Kennedy disease (KD) antibody
    • NR3C4 antibody
    • Nuclear receptor subfamily 3 group C member 4 (NR3C4) antibody
    • Nuclear receptor subfamily 3 group C member 4 antibody
    • SBMA antibody
    • SMAX1 antibody
    • Spinal and bulbar muscular atrophy (SBMA) antibody
    • Spinal and bulbar muscular atrophy antibody
    • Testicular Feminization (TFM) antibody
    • TFM antibody
    see all

Images

  • Immunohistochemistry (Formaline/PFA-fixed paraffin-embedded sections) analysis of human prostate tissue labeling Androgen Receptor with ab3509 at 1/20 (right). Antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 minutes. Tissues were blocked in 3% H2O2-methanol for 15 minutes at room temperature. A HRP-conjugated secondary antibody was used. Left - negative control.

References

This product has been referenced in:
  • Casati L  et al. Androgen receptor activation by polychlorinated biphenyls: epigenetic effects mediated by the histone demethylase Jarid1b. Epigenetics 8:1061-8 (2013). Read more (PubMed: 23907094) »
  • Bhattacharya I  et al. A switch in Sertoli cell responsiveness to FSH may be responsible for robust onset of germ cell differentiation during prepubartal testicular maturation in rats. Am J Physiol Endocrinol Metab 303:E886-98 (2012). WB, ICC/IF ; Rat . Read more (PubMed: 22850685) »
See all 3 Publications for this product

Customer reviews and Q&As

1-10 of 11 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Plethodontid Salamander Tissue sections (testes)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Unitrieve
Permeabilization
No
Specification
testes
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 75% · Temperature: 4°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Sep 28 2015

Application
Western blot
Loading amount
60 µg
Gel Running Conditions
Reduced Denaturing (12)
Sample
Human Cell lysate - whole cell (LNCaP)
Specification
LNCaP
Blocking step
Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 26°C

Abcam user community

Verified customer

Submitted Jan 13 2015

Application
Western blot
Loading amount
60 µg
Gel Running Conditions
Reduced Non-Denaturing (Native) (12)
Sample
Mouse Cell lysate - whole cell (Mouse Testis)
Specification
Mouse Testis
Blocking step
(agent) for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 26°C

Dr. Francesco Elia Marino

Verified customer

Submitted Mar 21 2014

Answer

Thank you for your reply.

I have had a look at ab9474 and the immunogen used to raise this antibody has been taken from residues 299-315 of human AR (SwissProt reference http://www.uniprot.org/uniprot/P10275). We do not have any information as to the cross reactivity of this antibody with the macaque protein (SwissProt reference http://www.uniprot.org/uniprot/Q6QT55)but due to this sequence homology (100%, see attachment) we would expect the antibody to be able to detect it. However, as we have not tested it ourselves we cannot be sure. It would definitly be worth trying and we would be very interested to know how you get on. If you'd like to share your findings (whether positive or negative) you can earn Abpoints by submitting Abreviews of your results. This should only take 5-10 minutes and the reward points can be used to redeem Amazon vouchers or claim the value off future Abcam orders. More information on this scheme can be found form the following link:

https://www.abcam.com/abreviews

As requested I have arranged for the ab9474 to be sent to you. This is on order number1095362 (purchase order number FOCR JB12-1218). This should reach you tomorrow. If you have any problems receiving it please do let me know. I would also be very interested to hear how you get on with the replacement antibody.

Until then, I wish you all the best with your research.

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Question

Product code: 3509
Lot number: GR64020-1
Inquiry: Replacement or refund for product
1) Abcam product code ab3509
2) Abcam order reference number or product batch number GR64020-1
3) Description of the problem: The antibody does not detect human AR. High backgrownd
4) Sample preparation: Type of sample: whole cell lysates C4-2 prostate cancer cells Lysis buffer: RIPA Protease inhibitors: cocktail Phosphatase inhibitors: yes Reducing agent: yes Boiling for ≥5 min? yes Protein loaded 30ug/lane Positive control: two other AR antibodies Negative control: siRNA anti-AR transfected cells
5) Percentage of gel: 4-12% Type of membrane: PVDF Protein transfer verified: yes Blocking agent and concentration: 5%milk PBS-T Blocking time: 2h Blocking temperature: RT
6) Primary antibody (If more than one was used, describe in “additional notes”) : Concentration or dilution: 1/500 and 1/1000 Diluent buffer: PBS-T 1% milk Incubation time: 12h Incubation temperature: 4°
7) Secondary antibody: GAR HRP Concentration or dilution: 1:10000 Diluent buffer: PBS-T 1% milk Incubation time: 1h Incubation temperature: RT Fluorochrome or enzyme conjugate: HRP
8) Washing after primary and secondary antibodies: Buffer: PBS-T Number of washes: 3
9)Detection method: luminesence SuperSignal West Dura
10) How many times have you run this staining? 3 Do you obtain the same results every time? yes What steps have you altered to try and optimize the use of this antibody? concentration of primary antibody, type of sample (mouse tissu)

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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 moresuccessful.

Having reviewed the protocol details, and the conclusive evidence provided with the other two antibodies used, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial. I am very sorry for the inconvenience this has caused you andwould be pleased to arrange for a free of charge replacement, credit note, or refund in compensation.

Having had a look at the alternatives from our catalogue I would like to offer you the mouse monoclonal https://www.abcam.com/Androgen-Receptor-antibody-AR-441-ab9474.htmlor the rabbit polyclonal https://www.abcam.com/Androgen-Receptor-antibody-ChIP-Grade-ab74272.html. Both of these antibodies have been tested in the application you are using as well as the species of sample the protein being detected derives from. If you would like for me to have this sent to you could you please confirm the order number (or purchase order, or the approximate date of delivery and delivery address).

Alternatively, if you would prefer an antibody against a different target please do let me know.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

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Answer

Thank you for your interest. It is very likely that ab9474 works in mouse but we have no data yet. According to Blast search, the homology between human and mouse AR is very high. http://www.uniprot.org/blast/uniprot/2011101050RXMELAA8 Furthermore, we have some alternative antibodies against AR in our catalogue which proved to recognize mouse AR: ab2742, ab74272, ab3509, ab52615 Normal brain, epididymis, prostate, seminal vesicle; or skin and prostate cancer can be used as good positive control. I hope this helps and if I can assist further, please do not hesitate to contact me.

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Question

Thank you for your mail. Here, the customer filled with Abcam's questionnaires: WB and IP. Please check the attachments. Look forward to hearing from you for your suggestions. Thanks. If you have any questions, don't hesitate to contact with me please! WB questionaire>> 1. Order details: • Batch number: • Abcam order or Purchase order number: AB3509 • Antibody storage conditions (temperature/reconstitution etc):-20℃,aliquoted 2. Please describe the problem (high background, wrong band size, more bands, no band etc). more bands 3. On what material are you testing the antibody in WB? • Species: LNCap cell line • Cell extract or Nuclear extract: cell extract • Purified protein or Recombinant protein:cell lysate 3. The lysate • How much protein was loaded: 80-100ug • What lysis buffer was used:Tritonx-100 1ml, sodium desoxycholate 0.5g, SDS 0.1g, to total volume 100ml with PBS • What protease inhibitors were used:1mM PMSF • What loading buffer was used: 4×loading buffer for polyacrylamide SDS -PAGE • Did you heat the samples: temperature and time:100℃,boiled,5 minutes 4. Electrophoresis/Gel conditions/ Transfer conditions • Reducing or non reducing gel: polyacrylamide SDS-PAGE • Gel percentage : 8% • Transfer conditions: 3h, 4℃,with transfer electrophoresis apparatus 5. Blocking conditions • Buffer:TBST • Blocking agent: milk, BSA, serum, what percentage:5% dried skim milk • Incubation time:1h • Incubation temperature: room temperature 6. Primary Antibody • Specification (in which species was it raised against):rabbit anti human • At what dilution(s) have you tested this antibody: 1:500, 1:1000 and 1:1500 • What dilution buffer was used: 5% dried skim milk in TBST • Incubation time:overnight • Incubation temperature:4℃ • What washing steps were done: wash with TBST buffer,shaking, 3 times, 10min every time 7. Secondary Antibody • Specification (in which species was it raised against)?goat anti rabbit IgG • At what dilution(s) have you tested this antibody:1:2000 • Incubation time: ,50min, room temperature • Wash steps: wash with TBST buffer,shaking, 3 times, 10min every time • Do you know whether the problems you are experiencing come from the secondary?No 8. Detection method ECl+ 9. Background bands • Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): YES • Is the blocking step sufficient? YES • Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) YES • At what size are the bands migrating? Could they be degradation products of your target? 110kd • Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 11. Did you apply positive and negative controls along with the samples? Or documents ?Please specify. LNCap cell line express AR protein,it must be positive.we didn’t do negative control. 10. Optimization attempts • How many times have you tried the Western?3 times • Do you obtain the same results every time e.g. are background bands always in the same place? YES • What steps have you altered? Primary Antibody dilution concentration, refer to the above item. IP questionaire>> 1. Order details: The same as the WB questionnaire. 2. Please describe the problem (high background, wrong band size, more bands, no band etc). no band 3. Did you test the antibody in WB with a positive control Yes 4. On what material are you testing the antibody in IP • Species: • Cell extract or Nuclear extract: Cell extract • Purified protein or Recombinant protein: 5. The lysate • What lysis buffer was used: RIPA Buffer • What protease inhibitors were used: Leupeptin, Aprotinin, Pepstatin and PMSF • What detergent: NP40, SDS, other : NP40 6. The Immunoprecipitation step How did you immunoprecipitate: Using Staph. Aureus, Protein A Sepharose, Protein G, Protein A/G, Or a second antibody Protein A Sepharose Antibody-lysate incubation: • How much lysate was immunoprecipitated: 400μl , 2μg/μl • How much antibody was added to the lysate: 10μl • How long did you leave the antibody and lysate to immunoprecipitate: Overnight(14 h) • Temperature: 4℃ • Was this done under agitation: Yes Addition of Protein-A/G beads: • Did you pre-block the beads: Yes • How much beads did you add: 100μl(50%) • How long did you incubate the beads with the lysate-antibody mix for:5 h • What temperature: 4℃ Addition of the loading buffer: • What loading buffer was used: 2 x sample buffer(contain SDS) • Did you heat the samples: temperature and time: 100℃,5 minutes 7. Western blotting conditions • Did you load a positive control: Yes 8. Secondary Antibody • Specification (in which species was it raised against)? Goat • At what dilution(s) have you tested this antibody:1:2000 • Incubation time: Room temperature for 50 minutes • Wash step:1╳ PBS, three times ,10 min for every time • Do you know whether the problems you are experiencing come from the secondary? Yes, there is no problem with the secondary antibody. 9. Detection method ECl+ 10. Background bands • Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) Yes, multiple short washes • At what size are the bands migrating? 110KD • Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 11. Did you apply positive and negative controls along with the samples: Please specify: yes, I had applied the total cell extract for positive. 12. Optimization attempts • How many times have you tried the IP? One time • Do you obtain the same results every time e.g. are background bands always in the same place? Yes • What steps have you altered? None.

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Answer

Thank you for providing the customer's full IP and western blotting protocols. Both protocols are very good and I have only a few questions I hope you can clarify: -has the customer tried WB with the RIPA buffer? The WB questionnaire mentions a trionx100 buffer, but the IP questionnaire mentions RIPA, a more suitable buffer to extract nuclear proteins like the androgen receptor. -has the customer tried WB with buffer containing protease inhibitors other than PMSF? The IP questionnaire mentions leupeptin, aprotinin, pepstatin, and PMSF. I think this is a very good cocktail and am concerned that if these inhibitors were not used, the lower bands are due to degradation of the androgen receptor protein. -Has the customer tried incubating more dilute antibody in WB? I tried to compare the images from WB and IP and was unclear as to whether lane 1 of IP is the same sample as the WB samples (LNCap cell lysate). If so, it is strange that the IP image does not show the 110kDa band, can the customer clarify what lysate was run in lane 1? Thank you for providing these few more informations, I really appreciate these clarifications to better understand the customer's problems.

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Answer

Thank you for contacting us for technical support with ab3509. The fact that the customer does not have a lot number is not a problem, we are always happy to help our customers and will try hard to help them, even without a lot number. I would recommend to collect the full IP and WB protocol from your customer, using our questionnaires, once we receive them we aim to reply in 24 hours with an answer. Thank you for asking those details to the customer, I hope we will be able to resolve the problem for them.

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Question

BATCH NUMBER 178261 ORDER NUMBER 147 DESCRIPTION OF THE PROBLEM Non-specific staining SAMPLE Adult rat brain tissues PRIMARY ANTIBODY Abcam rabbit anti-androgen receptor, 1:100-1:300 overnight at 4°C DETECTION METHOD ABC method POSITIVE AND NEGATIVE CONTROLS USED Positive control: rabbit anti-tyrosine hydroxylase antibody ->worked nicely. Negative control: omitted the primary antibody->no signal ANTIBODY STORAGE CONDITIONS Aliquoted into 10 ul, and stored at -20°C freezer FIXATION OF SAMPLE 4% paraformaldehyde and 4 h postfixation ANTIGEN RETRIEVAL We had tried with/without the antigen retrieval (heat in citric acid) PERMEABILIZATION STEP 0.025% Triton X BLOCKING CONDITIONS 10% normal goat serum in TBS for 2 h at RT SECONDARY ANTIBODY [another company] biotinylated goat anti-rabbit, 1:200 for 2h at RT HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Dilution factors of the primary antibody ADDITIONAL NOTES I have bought many antibodies from abcam and was satisfied with your products. So I changed androgen receptor antibody from [another company] to abcam eventhough I got a good result with [another company] antibody. Unfortunately, I could not get a good result with your product. As my data which I attach, I used same sample for androgen receptor antibody and tyrosine hydroxylase antibody but I got a clear data with tyrosine hydroxylase antibody while there is non specific signal with your product. Infact, androgen receptor is expressed in the nuclear thus the signal is supposed to be only in the nuclear.

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Answer

Thank you for the image you recently provided and for the customer's protocol details, it enabled me to understand the problem very rapidly. The customer's protocol is very good and similar to the one we recommend. The customer does not mention whether endogenous peroxidases were quenched and has not used picric acid in the perfusion solution, a fixative which was used with PFA when testing ab3509 in IHC. We were able to source the protocol with this product and were informed to not use H2O2 pretreatment as this eliminated the nuclear staining. On the datasheet is also a detailed protocol for IHC with ab3509. As this information was not available at the time of purchase, if the customer is unhappy with using tissue fixed in PFA-picric acid or is still experiencing problems with such a fixation method I can offer him/her a credit note, Thank you for forwarding these comments to your customer,

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Answer

This antibody is a rabbit polyclonal made using the recombinant full length protein from human. Being a polyclonal, it will therefore recognise a number of epitopes across the whole protein.

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1-10 of 11 Abreviews or Q&A

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