Invitrogen

Phospho-IRS1 (Ser616) Recombinant Rabbit Monoclonal Antibody (26H8L6)

Advanced Verification

This Antibody was verified by Cell treatment to ensure that the antibody binds to the antigen stated.

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Protocols

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Phospho-IRS1 (Ser616) Antibody in Immunocytochemistry (ICC/IF) — Immunofluorescence was performed on MCF7 cells treated with IGF1 (100 ng/mL/20 minutes). These cells were permeabilized and fixed for detection of IRS1 (pS616) using Anti-IRS (pS616) Recombinant Rabbit Monoclonal Antibody (Product # 701748, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 0.4 µg/mL, 1:2500). Panel a) shows representative cells that were stained for detection and localization of IRS (pS616) protein (green), Panel b) is stained for Nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938, 1:50). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 594 Phalloidin (Product # A12381, 1:200). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of IRS (pS616). Panel e) represents cells without treatment and panel f) represents control cells with no primary antibody to assess background.

Phospho-IRS1 (Ser616) Antibody in Western Blot (WB) — A) Western blot analysis was performed on whole cell extracts (30 µg lysate) of MCF7 (Lane 1), MCF7 treated with Insulin (100nM/10 min) (Lane 2), L6 (Lane 3), L6 treated with Insulin (100nM/10 min) (Lane 4), C2C12 (Lane 5) and C2C12 treated with Insulin (100nM/10 min) (Lane 6). The blots were probed with Anti-IRS1 (pS616) Recombinant Rabbit Monoclonal Antibody (Product # 701748, 1-3 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Bands at 65, 130, 180 kDa were observed as IRS is known to have multiple isoforms, across cell lines according to the treatment. B) To confirm specificity, competition was performed with the phosphopeptide (10 µg/mL) as shown on the right. The peptide competes with the antibody and prevents it from binding to the target protein. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane by overnight wet transfer. The membrane was probed with primary and secondary antibody following blocking with 5% nonfat milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Phospho-IRS1 (Ser616) Antibody — Altered expression of proteins upon cell treatment demonstrates antibody specificity. Immunofluorescence analysis of IRS1 [pS616] using IRS1 [pS 616] Recombinant Rabbit Monoclonal Antibody (Product # 701748) shows induction of phosphorylation of IRS1 in MCF7 cells upon treatment with IGF1. {TM}

Product Details

701748

Applications	Tested Dilution	Publications
Western Blot (WB)	1-3 µg/mL	-
Immunocytochemistry (ICC/IF)	1 µg/mL	-

Product Specifications
Species Reactivity	Human
Host/Isotype	Rabbit / IgG
Class	Recombinant Monoclonal
Type	Antibody
Clone	26H8L6
Immunogen	Phosphopeptide corresponding to Human IRS1 Ser616 (aa 612-619) View immunogen
Conjugate	Unconjugated
Form	Liquid
Concentration	0.5 mg/mL
Purification	Protein A
Storage buffer	PBS, pH 7.2
Contains	0.09% sodium azide
Storage conditions	Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
RRID	AB_2532503

Product Specific Information

This antibody is predicted to react with Monkey, Bovine, Horse and Cat.

Recombinant rabbit monoclonal antibodies are produced using in vitro expression systems. The expression systems are developed by cloning in the specific antibody DNA sequences from immunoreactive rabbits. Then, individual clones are screened to select the best candidates for production. The advantages of using recombinant rabbit monoclonal antibodies include: better specificity and sensitivity, lot-to-lot consistency, animal origin-free formulations, and broader immunoreactivity to diverse targets due to larger rabbit immune repertoire.

Target Information

IRS-1, a major substrate of the insulin receptor, is phosphorylated in response to stimulation of cells by insulin, insulin-like growth factor 1 (IGF-1) and interleukin 4 (IL-4). IRS-1 is phosphorylated on serine, threonine and tyrosine residues in a variety of tissues. An insulin-sensitive serine/threonine kinase casein kinase II mediates a portion of the insulin-stimulated serine/threonine phosphorylation of overexpressed IRS-1 in vivo. Thr 502 is identified as the major casein kinase II-catalyzed phosphorylation site in rat IRS-1, and Ser 99 is an additional phosphorylation site catalyzed by casein kinase II. Thus, casein kinase II-catalyzed phosphorylation of IRS-1 may be a component of the intracellular insulin signaling cascade. IRS-1 contains three putative binding sites for 14-3-3 (Ser 270, Ser 374 and Ser 641) and the motif around Ser 270 is located in the phosphortyrosine binding domain of IRS-1, which is responsible for the interaction with the insulin receptor.

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

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Bioinformatics

Protein Aliases: Insulin receptor substrate 1; IRS-1

Gene Aliases: HIRS-1; IRS1

UniProt ID: (Human) P35568

Entrez Gene ID: (Human) 3667

Function(s)

Process(es)

signal transducer activity transmembrane receptor protein tyrosine kinase adaptor activity protein kinase C binding Ras guanyl-nucleotide exchange factor activity insulin receptor binding insulin-like growth factor receptor binding protein binding 1-phosphatidylinositol-3-kinase activity SH2 domain binding phosphatidylinositol 3-kinase binding phosphatidylinositol-4,5-bisphosphate 3-kinase activity

Phospho-IRS1 (Ser616) Recombinant Rabbit Monoclonal Antibody (26H8L6)

Product Details

701748

Western Blot (WB)

Immunocytochemistry (ICC/IF)

Species Reactivity

Host/Isotype

Class

Type

Clone

Immunogen

Conjugate

Form

Concentration

Purification

Storage buffer

Contains

Storage conditions

RRID

Product Specific Information

Target Information

References (0)

Bioinformatics

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