Anti-Insulin Receptor (phospho Y972) antibody (ab5678)
Key features and details
- Rabbit polyclonal to Insulin Receptor (phospho Y972)
- Suitable for: ICC/IF, WB
- Reacts with: Human, Chinese hamster
- Isotype: IgG
Overview
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Product name
Anti-Insulin Receptor (phospho Y972) antibody
See all Insulin Receptor primary antibodies -
Description
Rabbit polyclonal to Insulin Receptor (phospho Y972) -
Host species
Rabbit -
Specificity
In some cell systems ab5678 has been shown to cross-react with IGF1R pY950 (75% homologous). -
Tested Applications & Species
See all applications and species dataApplication Species ICC/IF HumanWB Chinese hamster
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Immunogen
Synthetic peptide (Human). Synthetic phosphopeptide derived from the region of the human Insulin Receptor that contains tyrosine 972 (as numbered according to Ebina, et al. (tyrosine 960 according to Ullrich, et al.).
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Positive control
- IF: Insulin treated MCF7 cells. WB: CHO-T (Chinese hamster ovary cell line) cells transfected with a vector encoding the human insulin receptor and stimulated with insulin.
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General notes
Biological actions of insulin are mediated by the Insulin Receptor (IR), a receptor tyrosine kinase that regulates multiple signaling pathways through activation of a series of phosphorylation cascades. The IR is a heterotetrameric protein consisting of two ligand-binding alpha subunits and two beta subunits that each contain a tyrosine kinase domain. Insulin binding to the extracellular domain leads to autophosphorylation of the receptor and activation of the intrinsic tyrosine kinase activity, which allows appropriate substrates to be phosphorylated. Tyrosine 972 is in the juxtamembrane Asn-Pro- Glu-Tyr (NPEY) motif. Phosphorylation of IR tyrosine 972 is required for the binding and/or phosphorylation of the adapter protein Shc, the PTB domain, IRS-1, PI3 kinase, and the Suppressor of Cytokine Signaling (SOCS).
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.30
Preservative: 0.05% Sodium azide
Constituents: PBS, 1% BSA, 50% Glycerol -
Concentration information loading... -
Purity
Immunogen affinity purified -
Purification notes
The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated Insulin Receptor (IR). The final product is generated by affinity chromatography using an IR-derived peptide phosphorylated at tyrosine 972. -
Primary antibody notes
Biological actions of insulin are mediated by the Insulin Receptor (IR), a receptor tyrosine kinase that regulates multiple signaling pathways through activation of a series of phosphorylation cascades. The IR is a heterotetrameric protein consisting of two ligand-binding alpha subunits and two beta subunits that each contain a tyrosine kinase domain. Insulin binding to the extracellular domain leads to autophosphorylation of the receptor and activation of the intrinsic tyrosine kinase activity, which allows appropriate substrates to be phosphorylated. Tyrosine 972 is in the juxtamembrane Asn-Pro- Glu-Tyr (NPEY) motif. Phosphorylation of IR tyrosine 972 is required for the binding and/or phosphorylation of the adapter protein Shc, the PTB domain, IRS-1, PI3 kinase, and the Suppressor of Cytokine Signaling (SOCS). -
Clonality
Polyclonal -
Isotype
IgG -
Research areas
Images
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Immunocytochemistry/ Immunofluorescence - Anti-Insulin Receptor (phospho Y972) antibody (ab5678)
Immunofluorescence analysis of insulin treated MCF7 cells labelling Insulin Receptor (phospho Y972) (Panel a: green) using ab5678 at 2µg/mL in 1% BSA for 3 hours at room temperature, followed by Alexa Fluor 488® Goat Anti-Rabbit IgG Secondary Antibody at 1/400 dilution for 30 minutes at room temperature. Panel b:Nuclei were stained with DAPI (blue). Panel c: F-actin was stained with Alexa Fluor 594® Phalloidin (red). Panel d: Merged image showing membrane localization. Panel e: Untreated MCF7 cells. Panel f: Control, no primary antibodyl. The images were captured at 20X magnification.
Prior antibody incubation, MCF7 (human breast adenocarcinoma cell line) cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature, followed by treatment with 100nM of insulin for 5 min. Assay was done on 70% confluent log phase MCF7 cells.
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Western blot - Anti-Insulin Receptor (phospho Y972) antibody (ab5678)All lanes : Anti-Insulin Receptor (phospho Y972) antibody (ab5678) at 1/1000 dilution (2 hours at room temperature in a 3% BSA-TBST buffer)
Lane 1 : Unstimulated (-), CHO-T transfected with insulin receptor containing vector whole cell extract with 5% BSA-TBST buffer for one hour at room temperature
Lanes 2-5 : Stimulated (+) with 50 nM insulin for 5 minutes, CHO-T transfected with insulin receptor containing vector whole cell extract with 5% BSA-TBST buffer for one hour at room temperature
Secondary
All lanes : Goat F (ab')2 anti-rabbit IgG HRP conjugateUpregulation and Antibody-Peptide Competition:
Prior primary antibody incubation:
1 and 2 - no peptide;
3 - non-phosphorylated peptide corresponding to the phosphopeptide immunogen;
4 - generic phosphotyrosine-containing peptide;
5 - phosphopeptide immunogen.
SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF.
The data show that only the phosphopeptide corresponding to ab5678 completely blocks the antibody signal, demonstrating the specificity of the antibody.
The data also show up-regulation of the signal upon stimulation with insulin in this cell system.
