Technical guide & protocol Tech Support

Product Specifications

Analytical sensitivity

<1 U/mL

Assay range

1.6-100 U/mL

Sample type/volume

Cell Lysate
10 µL

Hands-on time

1 hr 20 min

Time-to-result

4 hr

Homogenous (no wash)

No

Interassay CV

7.4%

Intraassay CV

5.8%

Instrument

Colorimetric Microplate Reader

Product size

96 Tests

Contents

Pre-coated 96 well plate
Standard
Standard Dilution Buffer
Detection Antibody
anti-rabbit-HRP
HRP Diluent
Wash Buffer
Chromogen
Stop Solution
Adhesive Plate Covers

Shipping conditions

Wet ice

Storage

2-8°C

Protein name

IRS1

Target site-specificity

Phosphorylated Ser312

Protein subtype

Adaptor Proteins

Protein family

Other Proteins

Species (tested)

Human, Mouse, Rat

Assay kit format

Sandwich ELISA Kit

Detector antibody conjugate

HRP

Label or dye

HRP

About This Kit

The Human IRS1 (Phospho) [pS312] ELISA Kit is a solid-phase sandwich Enzyme-Linked Immunosorbent Assay (ELISA) designed to detect and quantify the level of IRS1 (Phospho) [pS312] in fresh or frozen human cell lysates. Cross-reactivity has been observed in mouse and rat cells. The assay recognizes both natural and recombinant IRS1 (Phospho) [pS312].

Principle of the method

A monoclonal capture antibody specific for IRS1 (Phospho) [pS312] has been coated onto the wells of the 96-well plate. During the first incubation, standards of known content and unknown samples are pipetted into the wells and the antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for the target protein is added to the wells and serves as a detection antibody by binding to the immobilized protein captured during the first incubation. After washing, a horseradish peroxidase labeled anti-rabbit IgG is added. This binds to the detection antibody to complete the four member sandwich. After a third incubation and washing to remove all the unbound enzyme, a substrate solution (TMB) is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of target protein present in the original specimen and the optical density can be read on a standard microplate reader.

Rigorous validation

Each manufactured lot of this ELISA kit is quality tested for criteria such as sensitivity, specificity, precision, and lot-to-lot consistency. See manual for more information on validation.

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.

Bioinformatics

Gene aliases : G972R, HIRS-1, IRS-1, IRS1, IRS1IRM

Gene ID : (Human) 3667, (Rat) 25467, (Mouse) 16367

Gene symbol : IRS1, Irs1, Irs1

Protein Aliases : Insulin receptor substrate 1, IRS-1, pp185

UniProt ID (Human) P35568, (Rat) P35570, (Mouse) P35569

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