Anti-Insulin Receptor beta 抗体 [18-44] (ab983)

Reviews (1)Q&A (6)References (5)

製品の概要

  • 製品名
    Anti-Insulin Receptor beta antibody [18-44]
    Insulin Receptor beta 一次抗体 製品一覧
  • 製品の詳細
    Mouse monoclonal [18-44] to Insulin Receptor beta
  • 由来種
    Mouse
  • 特異性
    This antibody reacts specifically with the beta subunit of the insulin receptor.
  • アプリケーション
    適用あり: Flow Cyt, ICC/IF, Blocking, Functional Studies, IP, WBmore details
  • 種交差性
    交差種: Rabbit, Cow, Human
  • 免疫原

    Human placental insulin receptor.

  • 特記事項

    This product was changed from ascites to tissue culture supernatant on 19/12/2018. Please note that the dilutions may need to be adjusted accordingly. If you have any questions please do not hesitate to contact our scientific support team.

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab983 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
Flow Cyt Use 1µg for 106 cells.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

 
ICC/IF Use at an assay dependent concentration. PubMed: 18411068
Blocking Use at an assay dependent concentration. Dilute in PBS or medium which is identical to that used in the assay system.
Functional Studies Use at an assay dependent concentration. Inhibition of insulin binding: IM-9 lymphocytes = 35%, Adipocytes = 90% Concentration for half maximal effect: Inhibition of insulin binding = 0.2nM, Stimulation of lipogenesis = 0.3nM. This antibody has insulin-like biological activity and therefore activates the receptor kinase.
IP Use at an assay dependent concentration.
WB Use at an assay dependent concentration.

ターゲット情報

  • 関連性
    Insulin receptor mediates the biological activities of insulin by regulating multiple signaling pathways through activation of a series of phosphorylation cascades. The human insulin receptor is a heterotetrameric membrane glycoprotein consisting of disulfide-linked subunits in a ß-a-a-ß configuration. The ß-subunit (95kDa) possesses a single transmembrane domain with tyrosine kinase acivity, whereas the a-subunit (135kDa) is completely extracellular. The alpha subunits each contain insulin binding sites and are entirely extracellular in localization. The beta subunits each possess an extracellular domain, a single transmembrane domain, and a cytoplasmic tyrosine kinase domain. Binding of insulin to the alpha subunits induces a conformation change in the receptor which activates the kinase domain, stimulating tyrosine autophosphorylation of the receptor and tyrosine phosphorylation of at least five different insulin receptor substrates designated IRS-1-4, and Shc.
  • 細胞内局在
    Membrane; Single pass type I membrane protein.
  • 参照データベース
    • 別名
      • CD220 antibody
      • HHF5 antibody
      • HIR B antibody
      • INSR antibody
      • Insulin receptor antibody
      • Insulin receptor subunit beta antibody
      • IR antibody
      see all

    画像

    • Flow Cytometry - Anti-Insulin Receptor beta antibody [18-44] (ab983)
      Flow Cytometry - Anti-Insulin Receptor beta antibody [18-44] (ab983)

      Overlay histogram showing HepG2 cells stained with ab983 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab983, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

      This image was generated using the ascites version of the product. 

    • Immunocytochemistry/ Immunofluorescence - Anti-Insulin Receptor beta antibody [18-44] (ab983)
      Immunocytochemistry/ Immunofluorescence - Anti-Insulin Receptor beta antibody [18-44] (ab983)Image from Thiel CT et al., Mol Genet Metab. 2008 Jul;94(3):356-62. Epub 2008 Apr 14. Fig 4.; doi:10.1016/j.ymgme.2008.02.013; 14 April 2008, Molecular Genetics and Metabolism, 94 (3), 356–362, with permission from Elsevier.

      ab983, staining Insulin Receptor beta in Human skin fibroblast cells, by Immunocytochemistry/ Immunofluorescence.

      Fibroblasts cultured from skin biopsies of a patient with Rabson–Mendenhall syndrome and a healthy control, were fixed in methanol. Samples were fixed with primary antibody and a FITC-labeled donkey polyclonal mouse antibody (ab7057) was used as a secondary antibody. DNA was counterstained with DAPI.

      This image was generated using the ascites version of the product. 

    参考文献

    This product has been referenced in:
    • Yosten GL  et al. Evidence for an interaction between proinsulin C-peptide and GPR146. J Endocrinol 218:B1-8 (2013). ICC/IF . Read more (PubMed: 23759446) »
    • Thiel CT  et al. Two novel mutations in the insulin binding subunit of the insulin receptor gene without insulin binding impairment in a patient with Rabson-Mendenhall syndrome. Mol Genet Metab 94:356-62 (2008). ICC/IF ; Human . Read more (PubMed: 18411068) »
    See all 5 Publications for this product

    ab983 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

    レビューと Q&A

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    1-7 of 7 Abreviews or Q&A

    Western blot abreview for Anti-Insulin Receptor beta antibody [18-44]

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Loading amount
    15 µg
    Gel Running Conditions
    Non-reduced Denaturing (10)
    Sample
    Mouse Tissue lysate - other (Hep G2)
    Specification
    Hep G2
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
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    Abcam user community

    Verified customer

    投稿 Jan 27 2014

    Question


    I would like to ask if you offer the insulin receptor antibody specific
    for exon 11 (aa 745-756). The goal should be to distinguish between IR-A
    and IR-B isoform of insulin receptor. Exon 11 is specific for IR-B
    isoform. Or if you offer alternative how to "catch" specifically IR-B
    isoform of insulin receptor.
    Thank you very much.
    Yours sincerely,

    Read More
    Answer



    According to the Uniprot homepage the alpha chain is located from amino acid 28-758, whereas the beta chain is from aa 763-1382:

    http://www.uniprot.org/uniprot/P06213

    ab69508 has its immunogen in the cytoplasmic domain of the beta subunit; this sequence seen here http://www.uniprot.org/uniprot/P06213#PRO_0000016687 as aa 980-1382 is buried in the beta subunit and does not fall in the alpha subunit which is aa 28-758 so I would not expect cross reactivity with alpha in this case. This has not been experimentally tested.

    https://www.abcam.com/index.html?datasheet=69508 (or use the following: https://www.abcam.com/index.html?datasheet=69508).

    ab983 : I am happy to confirm that product ab983 is specific for the β-subunit of human insulin receptor. This was determined by (1) immunoblotting on human insulin receptor and (2) epitope analysis of short receptor peptides. The minimum epitope for this antibody was defined as a sequence PEEHRP corresponding to amino acids 765-770 close to the N-terminus of the β-subunit.

    https://www.abcam.com/index.html?datasheet=983 (or use the following: https://www.abcam.com/index.html?datasheet=983).

    ab80527 : This antibody was generated using synthetic peptide corresponding the aa Tyr-KKNGRILTLPRSNPS from the C-terminal of human insulin receptor B-subunit. And therefore is also expected to be specific for the beta chain.

    https://www.abcam.com/index.html?datasheet=80527 (or use the following: https://www.abcam.com/index.html?datasheet=80527).

    More information on these antibodies can be found on their respective datasheets by following the links.

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    Question

    Post complaint survey:
    ...
    Q. Could we have done anything to better resolve the problem?
    A.The original CD63 antibody did not work despite the trying various permutations of blocking reagents etc. I requested an alternative antibody TSG101 (ab983)and this was offered free of charge to compensate for the original CD63. The TSG101 was used on saliva samples and has been published. I managed to get a positive immunosignal however it was comparatively weak to the published data even though I had followed the paper protocol. The data sheet that accompanied the antibody did not have an immunoblotting protocol. Please could abcam suggest a protocol as the aliqout was small to start with so I would like to avoid repeating the immunoblotting. KInd regards xx
    Q. Would you mind if our scientific support team contacted you to better understand what happened and reach an agreeable solution?
    A.No, I would not mind
    ...

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    Answer

    Thank you very much for having provided your feedback in the last survey.

    I am happy to hear that you were satisfied with our service and that the ab83 does work better than the original antibody received. I am sorry though, that the ab83 still does not work to your entire satisfaction. The laboratory used a classical protocol with 5% milk blocking to test this antibody. Would you like to troubleshoot the protocol together? I am happy to look into troubleshooting the ab83. If you would like our help, please provide the additional information on the samples and the protocol used with an image inthe attached questionnaire.

    Thank you for your cooperation. Please do not hesitate to contact us, either with the information, or also if you have any other question or concern.

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    Question

    What is the epitope of this antibody? Do you know if it binds C-terminally?

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    Answer

    Thank you for your inquiry and your patience with this reply. We have limited data regarding the specific epitope for this antibody, but it appears that it is amino acids 765-770 (PEEHRP). I hope this information helps. Please contact us with any other questions.

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    Question

    How are you all, love your team, anyways i have a customer which used your product and all failed on WB & IP. She would like to receive credit on this, can you credit our account? below is the protocol I had her email to me as i know this is required for your team to see if followed procedure correctly. Please let me know ASAP! Thanks (word per word from customer) I used Cat. No.AB983, Lot #101002 both in an immunoprecipitation experiment and a Western blot. It did not work in either application. Immunoprecipitation Protocol (from another company) 1. Add 1 mg of cell lysate at a concentration of roughly 1µg/µl total cell protein to a microcentrifuge tube. 2. Pre-clean the lysate by adding 60µl (30µl packed beads) of washed Protein A agarose bead slurry (Catalog # 16-125). Gently rock at 4°C for 1 hour. 3. Collect the agarose beads by pulsing (5 seconds in the microcentrifuge at 14,000 x g), and collect the supernatant. 4. Added 10 µl of anti-Insulin Receptor, Ab983 to the supernatant. 5. Gently rock the reaction mixture at 4°C overnight. 6. Capture the immunocomplex by adding 60µl of washed Protein A agarose bead slurry (30µl packed beads). 7. Gently rock the reaction mixture at 4°C for 1-2 hours. 8. Collect the agarose beads by pulsing (5 seconds in the microcentrifuge at 14,000 x g), and drain off the supernatant. Wash the beads 4 times with ice-cold cell lysis buffer. 9. Resuspend the agarose beads in 60µl 2X Laemmli sample buffer. 10. boil at 95°C for 5 minutes. 11. Collect the beads after boiling using a microcentrifuge pulse. 12. Perform SDS-PAGE and immunoblot analysis on a sample of the supernatant fraction. WESTERN: Used AB983 at a 1:1000 concentration in BSA Binding Buffer. No detection. [Another company's ab worked great] I would love to receive credit on the credit card for this purchase. Thanks.

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    Answer

    Thanks for your email and I'm sorry to hear that your customer is experiencing difficulty with this antibody. I do require some additional details in order to investigate this. Exactly what type of samples were used? And from which species? How was the sample prepared? For the Western, how much protein was loaded? Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)? How long was the primary incubation period? What were the blocking conditions and for how long? What was the secondary antibody, concentration, and incubation period? Also, with the other company's antibody that was used - was it a Insulin Receptor beta antibody? Thank you and I look forward to hearing from you.

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    Question

    I want to know how good the reactivity is with bovine (cow) species as it was mentioned in the product data sheet. Could you please send me any western blot pictures or any other data showing the reactivity of this antibody in bovine species? Thank you in advance and hoping to hear from you at your earliest.

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    Answer

    Thank you for your enquiry. The reactivity of this antibody with bovine and rabbit insulin receptor is extremely weak. In Western blots, a very faint band can be seen in some samples, but this banding is not seen consistently in all samples. We mention this weak cross-reactivity in our data sheet in case this is potentially problematic for users of this antibody, but we do not recommend this antibody for detecting bovine insulin receptor.

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    Question

    What is the reactivity of the above antibody in bovine samples? On the datasheet it states "weak reactivity, but what does the WB look like and what dilution is needed. ?

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    Answer

    Thank you for your enquiry. This antibody produces very faint banding when used at relatively high concentration (25 ug/ml) to immunoblot bovine or rabbit insulin receptor. In contrast, immunoblotting on human insulin receptor produces a sharp, strong band. We do not recommend this antibody for detection of bovine insulin receptor, but we wanted to bring this weak level of cross-reactivity to customers' attention in case it could pose a problem in someone's assays.

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    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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