Invitrogen

Phospho-IR/IGF1R (Tyr1162, Tyr1163) Polyclonal Antibody

Advanced Verification

This Antibody was verified by Cell Treatment to ensure that the antibody binds to the antigen stated. View Details

4 Published Figures

15 References

View all (6) IR/IGF1R antibodies

Datasheet

Datasheet

Product Image Gallery

Phospho-IR/IGF1R (Tyr1162, Tyr1163) Antibody in Cell Treatment — Modulation of target protein phosphorylation by cell treatment demonstrates antibody specificity. Immunofluorescence analysis of IR/IGF1R pY1162pY1163 using IR/IGF1R (pY1162pY1163) polyclonal antibody (Product # 44-804G) shows induced expression of IR/IGF1R (pY1162pY1163) in the membrane of MCF7 cells treated with insulin. {TM}

Phospho-IR/IGF1R (Tyr1162, Tyr1163) Antibody in Western Blot (WB) — Upregulation and Antibody-Peptide Competition. Extracts of CHO-T cells over-expressing the human insulin receptor unstimulated (1) or stimulated with 100 nM insulin for 10 min at 37°C (2-5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4°C and incubated with the IR/IGF1R (pYpY1162/1163) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the phosphopeptide immunogen (2), the non-phosphorylated peptide corresponding to the phosphopeptide immunogen (3), or a generic phosphotyrosine-containing peptide (4). After washing, the membrane was incubated with goat F (ab')2 anti-rabbit IgG HRP-conjugate (Product # ALI4404) and signals were detected using the Pierce SuperSignal™method. The data show that only the phosphopeptide corresponding to IR/IGF1R (pYpY1162/1163) completely blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the up-regulation of this site upon stimulation with insulin in this cell system.

Phospho-IR/IGF1R (Tyr1162, Tyr1163) Antibody in Immunofluorescence (IF) — Immunofluorescence analysis of IR/IGF1R (pYpY1162/1163) was done on 70% confluent log phase MCF7 cells treated with insulin (100nM for 5 min). The 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. The cells were labeled with IR/IGF1R (pYpY1162/1163) Rabbit polyclonal Antibody (Product # 44-804G) at 2 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing membrane localization. Panel e shows untreated MCF7 cells. Panel f shows no primary antibody control. The images were captured at 20X magnification.

Phospho-IR/IGF1R (Tyr1162, Tyr1163) Antibody — Published figure using Phospho-IR/IGF1R (Tyr1162, Tyr1163) polyclonal antibody (Product # 44-804G)

Product Details

Tested Applications

Dilution

Immunocytochemistry (ICC)

1:100-1:500

Immunofluorescence (IF)

1:100-1:500

Immunohistochemistry (IHC)

Assay Dependent

Western Blot (WB)

Assay Dependent

Published Applications

Western Blot (WB)

See 13 publications below

Immunohistochemistry (IHC)

See 1 publication below

Immunohistochemistry (Paraffin) (IHC (P))

See 1 publication below

Product Specifications

Species Reactivity

Human

Published species

Human, Mouse, Rat

Host / Isotype

Rabbit / IgG

Class

Polyclonal

Type

Antibody

Immunogen

Synthetic phosphopeptide derived from the region of IR/IGF1R that contains tyrosines 1162 and 1163 of the human insulin receptor (IR)

Conjugate

Unconjugated

Form

Liquid

Purification

Antigen affinity chromatography

Storage buffer

Dulbecco's PBS, pH 7.3, with 1mg/mL BSA, 50% glycerol

Contains

0.05% sodium azide

Storage conditions

-20°C

RRID

AB_2533762

Product Specific Information

The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody reactive with non-phosphorylated insulin/insulin-like growth factor-1 receptor (IR/IGF1R). The final product is generated by affinity chromatography using an IR/IGF1R-derived peptide that is phosphorylated at tyrosines 1162 and 1163 (tyrosines 1135 and 1136 for IGF1R. The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of IR/IGF1R that contains tyrosines 1162 and 1163 of the human insulin receptor (IR) as numbered according to Ebina, et al. (1150 and 1151 according to Ullrich et al.). The corresponding residues in the IGF1R are 1135 and 1136. The sequence is conserved in mouse and rat for both the IR and IGF1R. Although exhibiting a preference for IR/IGF1R, this antibody has been shown by both peptide competition and protein blotting to react with other dual-phosphotyrosine motifs from proteins such as c-Met and Shc.

This antibody has been used in western blotting and previous lots of this antibody have been used in immunostaining. Positive controls used with this antibody in western analysis were Chinese hamster ovary cells transfected with a vector containing human insulin receptor (CHO-T) and stimulated with insulin, or 3T3-L1 cells stimulated with insulin.

Target Information

Biological actions of insulin and IGF-1 are mediated by their respective cell surface receptors, both of which are receptor tyrosine kinases that regulate multiple signaling pathways through activation of a series of phosphorylation cascades. The IR and IGF-1R are heterotetrameric proteins consisting of two ligand-binding alpha subunits, which are completely extracellular, and two beta subunits that span the membrane with an intracellular portion that each contain a tyrosine kinase domain. These disulfide-linked subunits are in a beta-alpha-alpha-beta configuration in the mature receptor. Insulin/IGF-1 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. These two receptors differ in sequence in regions that confer specificity for the designated ligand as well as in certain intracellular signaling domains. These differences allow insulin and IGF-1 to regulate different physiological functions through receptors that share a very similar structure. Phosphorylation sites that are unique to each receptor presumably play a key role in these signaling differences. The catalytic loops within the tyrosine kinase domains of the IR/IGF-1R contain a three tyrosine motif corresponding to tyrosines 1158, 1162, and 1163 (for the IR) and tyrosines 1131, 1135, and 1136 (for the IGF-1R). It is generally believed that autophosphorylation within the activation loop proceeds in a processive manner initiating at the second tyrosine (1162 or 1135), followed by phosphorylation at the first tyrosine (1158 or 1131), then the last (1163 or 1136), upon which the IR or IGF-1R becomes fully active.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Bioinformatics

Protein Aliases: CD220; CD221; HHF5; IGF-I Receptor; IGF1 Receptor; IGF1R; IGFIR; IGFR; INSR; Insulin receptor; Insulin-like growth factor 1 receptor; Insulin-like growth factor I receptor; IR; JTK13; soluble IGF1R variant 1; soluble IGF1R variant 2

Gene Aliases: CD220; CD221; HHF5; IGF1R; IGFIR; IGFR; INSR; JTK13

UniProt ID: (Human) P08069, (Human) P06213

Entrez Gene ID: (Human) 3480, (Human) 3643

Function(s)