Anti-Insulin Receptor alpha antibody [83-7] (ab36550)

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Overview

  • Product name
    Anti-Insulin Receptor alpha antibody [83-7]
    See all Insulin Receptor alpha primary antibodies
  • Description
    Mouse monoclonal [83-7] to Insulin Receptor alpha
  • Host species
    Mouse
  • Specificity
    Does not cross react with the Human Type 1 IGF Receptor.
  • Tested applications
    Suitable for: Flow Cyt, IP, ELISA, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
    Does not react with: Rat
  • Immunogen

    Transfected 3T3 cells overexpressing the human Insulin Receptor alpha

  • Epitope
    ab36550 recognizes an epitope within amino acids 140-301 (the cysteine rich region)of the extracellular domain of the human Insulin Receptor alpha.
  • General notes


    The antibody enhances the binding of 125I insulin binding to the insulin receptor of HIR3.5/3T3 cells and stimulates insulin mediated 3H thymidine incorporation in these cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab36550 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
Flow Cyt Use 1µg for 106 cells.

(paraformaldehyde or methanol fixed cells)

 

 

 

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 
IP 1/5000.
ELISA 1/5000.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • Relevance
    The human insulin receptor is a heterotetrameric membrane glycoprotein consisting of disulfide linked subunits in a beta-alpha-alpha-beta configuration. The beta subunit (95 kDa) possesses a single transmembrane domain, whereas the alpha subunit (135 kDa) is completely extracellular. The insulin receptor exhibits receptor tyrosine kinase (RTK) activity. RTKs are single pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the gamma phosphate of ATP to tyrosine residues in protein substrates. RTKs are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. Included in this large protein family are the insulin receptor and the receptors for growth factors such as epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor. Receptor activation occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The interaction of insulin with the alpha subunit of the insulin receptor activates the protein tyrosine kinase of the beta subunit, which then undergoes an autophosphorylation that increases its tyrosine kinase activity. Three adapter proteins, IRS1, IRS2 and Shc, become phosphorylated on tyrosine residues following insulin receptor activation. These three phosphorylated proteins then interact with SH2 domain containing signaling proteins.
  • Cellular localization
    Membrane; single pass type I membrane protein.
  • Database links
    • Alternative names
      • CD220 antibody
      • HHF5 antibody
      • HIR A antibody
      • INSR antibody
      • Insulin receptor antibody
      • Insulin receptor subunit alpha antibody
      • IR antibody
      see all

    Images

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Insulin Receptor alpha antibody [83-7] (ab36550)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Insulin Receptor alpha antibody [83-7] (ab36550)
      ab36550 (1µg/ml) staining insulin receptor alpha in human pancreas using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of cytoplasm and basal cell membrane of proximal convoluted tubule cells.
      Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH 6.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
    • Flow Cytometry - Anti-Insulin Receptor alpha antibody [83-7] (ab36550)
      Flow Cytometry - Anti-Insulin Receptor alpha antibody [83-7] (ab36550)
      Overlay histogram showing Jurkat cells stained with ab36550 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab36550, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with methanol (5 min) used under the same conditions.

      Please note that Abcam do not have data for use of this antibody on non-fixed cells. We welcome any customer feedback.

    References

    This product has been referenced in:
    • Cieniewicz AM  et al. Novel Monoclonal Antibody Is an Allosteric Insulin Receptor Antagonist That Induces Insulin Resistance. Diabetes 66:206-217 (2017). Read more (PubMed: 27797911) »
    • Subramanian K  et al. Insulin receptor-insulin interaction kinetics using multiplex surface plasmon resonance. J Mol Recognit 26:643-52 (2013). Read more (PubMed: 24277609) »
    See all 6 Publications for this product

    Publishing research using ab36550? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-5 of 5 Abreviews or Q&A

    Question

    The labeling kit that I used is :

    Alexa Fluor 647 carboxylic acid, succinimidyl ester.

    I think there is something in the buffer which inhibits the labeling. I bought two vials from your company. After the labeling failed,

    I exchanged buffer to PBS and relabeled it. Now it is working. But I lost protein and time during the process. Right now, I do not have enough antibody. I hope you can give me at least one free vial.

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    Answer

    Thank you for providing these details. That is interesting, that the conjugation worked after the buffer exchange. The antibody is lyophilized from a PBS solution, and then reconstituted in PBS, according to the information I have. The solution you receive may be hypertonic compared to physiological saline, but I do not think that would affect the conjugation.

    I have issued a free-of-charge vial of ab36550. The delivery status is currently set to "customer pick-uip", which was the case for the previous order, but if you like, I can have it delivered. Just let me know. The order number is ****, for one vial of ab36550, anit-Insulin Receptor alpha. If you are picking up, you simply need to mention the order number at the reception desk in Building 200, 3rd floor.

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    Question

    Conjugation failed. Does the antibody contain any carrier protein? Is it purified?

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    Answer

    Thank you for brining this to our attention. I have confirmed that the antibody solution contains only purified antibody and PBS, with no carrier protein or preservatives. You mentioned that the Invitrogen conjugation kit successfully conjugated another antibody. Can you tell me the catalogue number of the kit? We have not had any complaints about the antibody, so I do not think it was degraded when we sent it. Do you know the PO number for your order?

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    Question

    Certificate of analysis for ab36550, order number ***.  

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    Answer

    Thank you for contacting us with your request. At Abcam, we are able to provide Certificates of Compliance instead of Certificates of Analysis, and I have attached the CoC for Ab36550 for the lot that you have purchased on order 982013.  Please let me know if you have any questions or need any additional information.  

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    Question

    Hi, Can you please let me know about the isoelectric point for this antibody "83-7" (ab36550). Thanks,

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    Answer

    Thank you for your enquiry and your interest in our products. I regret to inform you that the isoelectric point for this antibody has not been determined. Apologies! If you need any further assistance in the future, please do not hesitate to contact me.

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    Question

    I have a customer with the following enquiry: We have purchased another product off Abcam, and we would appreciate the molecular weight of the mouse monoclonal (83-7) to insulin receptor alpha (ab36550). Please let me know if you have this information available.

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    Answer

    Thank you for your enquiry. This product has yet to be tested in WB application therefore, we do not know the actual molecular weight. However, according to SwissProt database, the expected molecular weight should be around 156kDa. Please see the link below. http://br.expasy.org/cgi-bin/niceprot.pl?P06213 I hope this information will be helpful. If there is anything else that I can help you with, please do not hesitate to contact me.

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