General description
Insulin receptor substrate 1 (UniProt: P35568; also known as IRS-1) is encoded by the IRS1 gene (Gene ID: 3667) in human. The insulin receptor substrates (IRS-1, IRS-2, IRS-3, and IRS-4) are adaptor proteins involved in modulating cell growth, metabolism, survival, and differentiation by transducing receptor signaling to multiple downstream effectors. IRS family proteins contain a conserved N-terminal pleckstrin homology (PH) domain (aa 12-115 of human IRS-1), a phosphotyrosine-binding (PTB) domain (aa 160-264 of human IRS-1), and multiple tyrosine phosphorylation sites in the C-terminal region. The PTB domain mediates the interaction of IRS-1, -2 and -3 with IR juxtamembrane NPXYp motif, facilitating subsequent receptor-mediated IRS tyrosine phosphorylation. Upon phosphorylation, IRS proteins serve as binding sites for Src homology 2 (SH2) domain-containing proteins, such as SHP-2 and PI 3-kinase. Functions of IRS proteins are largely dependent on the phosphorylation of specific tyrosine residues, which in turn are subject to negative regulation by protein phosphatase activity. In addition, IRS proteins can be phosphorylated by serine/threonine kinases, such as Akt/PKB, GSK-3, mTOR, and PKC, and increased Ser/Thr phosphorylation reduces their binding to IR and tyrosine phosphorylation. When phosphorylated, tyrosine 612 is a pYMxM consensus motif PI3-kinase binding site, which leads to the activation of the PI3K/AKT signaling pathway. Inhibition of PI 3-kinase enhances insulin-stimulated IRS-1 Tyr612/Tyr941 phosphorylation (p85-binding sites) and increases IRS-1 association with PI 3-kinase p85 subunit. Serine phosphorylation of IRS1 is a involved in insulin resistance. Phosphorylation of serine 312 inhibits insulin action through disruption of IRS1 interaction with the insulin receptor. Defects in muscle IRS1 gene expression and function have been reported in insulin-resistant states, such as in obesity and type 2 diabetes. This ZooMAb® recombinant monoclonal antibody, generated by our propriety technology, offers significantly enhanced specificity, affinity, reproducibility, and stability over conventional monoclonals. (Ref.: Koh, A., et al. (2013). Mol. Cell Biol. 33(8); 1608-1620; Gao, Z., et al. (2002). J. Biol. Chem. 277(50); 48115-48121).
ZooMAb® antibodies represent an entirely new generation of recombinant monoclonal antibodies. Each ZooMAb® antibody is manufactured using our proprietary recombinant expression system, purified to homogeneity, and precisely dispensed to produce robust and highly reproducible lot-to-lot consistency. Only top-performing clones are released for use by researchers. Each antibody is validated for high specificity and affinity across multiple applications, including its most commonly used application. ZooMAb® antibodies are reliably available and ready to ship when you need them.
Immunogen
KLH-conjugated linear peptide corresponding to 12 amino acids surrounding phosphoserine 312 from human Insulin receptor substrate 1 (IRS1).
Application
Quality Control Testing
Evaluated by Western Blotting in lysates from CHO cells stably transfected with IR/IRS1 and treated with Calyculin A/Okadaic Acid.
Western Blotting Analysis: A 1:1,000 dilution of this antibody detected phospho-IRS1 (Ser312) in lysates from CHO cells stably transfected with IR/IRS1 and treated with Calyculin A (50 nM)/Okadaic Acid (500 nM) for 30 min.
Tested Applications
Western Blotting Analysis: 1:1,000 dilution of this antibody detected phospho-IRS1(Ser312) construct but did not react with non-phosphorylated construct.(Construct: Courtesy of Dr. Jesse Rinehart, Yale University, School of Medicine).
Immunocytochemistry Analysis: A 1:1,000 dilution from a representative lot detected p-IRS1(Ser312) in CHO cells stably transfected with IR/IRS1, serum starved, and treated with 50 nM Calyculin A and 500 nM Okadaic acid for 30 mins.
Peptide Inhibition Assay: Target band detection in lysate from CHO cells stably transfected with IR/IRS1 was prevented by preblocking of a representative lot with the immunogen phosphopeptide, but not the corresponding non-phosphopeptide.
Note: Actual optimal working dilutions must be determined by end user as specimens, and experimental conditions may vary with the end user.
Biochem/physiol Actions
Clone 6C20 is a ZooMAb® rabbit recombinant monoclonal antibody that specifically detects Insulin receptor substrate 1 (IRS1) phosphorylated on Serine 312.
Physical form
Purified recombinant rabbit monoclonal antibody IgG, lyophilized in PBS with 5% Trehalose, normal appearance a coarse or translucent resin. The PBS/trehalose components in the ZooMAb formulation can have the appearance of a semi-solid (bead like gel) after lyophilization. This is a normal phenomenon. Please follow the recommended reconstitution procedure in the data sheet to dissolve the semi-solid, bead-like, gel-appearing material. The resulting antibody solution is completely stable and functional as proven by full functional testing. Contains no biocide or preservatives, such as azide, or any animal by-products. Larger pack sizes provided as multiples of 25 µL.
Preparation Note
Recommend storage of lyophilized product at 2-8°C; Before reconstitution, micro-centrifuge vials briefly to spin down material to bottom of the vial; Reconstitute each vial by adding 25 µL of filtered lab grade water or PBS; Reconstituted antibodies can be stored at 2-8°C, or -20°C for long term storage. Avoid repeated freeze-thaws.
Reconstitute lyophilized antibody pellet with 25μL of ultrapure water or Phosphate Buffered Saline (PBS). Please refer to our reconstitution protocol and the specific application guidance on the suggested starting dilutions and sample type.
Legal Information
ZooMAb is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
| biological source | rabbit |
| Quality Level | 200 |
| recombinant | expressed in HEK 293 cells |
| conjugate | unconjugated |
| antibody form | purified antibody |
| antibody product type | primary antibodies |
| clone | 6C20, recombinant monoclonal |
| description | recombinant, expressed in HEK 293 cells |
| product line | ZooMAb® learn more |
| form | lyophilized |
| mol wt | calculated mol wt 131.59 kDa,observed mol wt ~165 kDa |
| purified by | using Protein A |
| species reactivity | rat, human |
| species reactivity (predicted by homology) | mouse |
| packaging | antibody small pack of 25 |
| enhanced validation | recombinant expression Learn more about Antibody Enhanced Validation |
| technique(s) | immunocytochemistry: suitable,inhibition assay: suitable,western blot: suitable |
| isotype | IgG |
| epitope sequence | N-terminal half |
| Protein ID accession no. | NP_005535.1 |
| UniProt accession no. | P35568 |
| shipped in | ambient |
| storage temp. | 2-8°C |
| Gene Information | human ... IRS1(3667) |
