IRS-1 (Phospho-Ser307) Fluorometric Cell-Based ELISA Kit

SKU:

FBCAB00018

Product Type:

ELISA Kit

ELISA Type:

Cell Based Phospho Specific

Reactivity:

Human

Mouse

Rat

Detection Method:

Fluorometric

Description

Description

IRS-1 (Phospho-Ser307) Cell-Based ELISA Kit

The IRS-1 (Phospho-Ser307) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor IRS-1 phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated IRS-1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on IRS-1 phosphorylation.

How does our IRS-1 (Phospho-Ser307) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of IRS-1 (Phospho-Ser307) concentration is achieved by an indirect ELISA format. In essence, IRS-1 (Phospho-Ser307) is captured by IRS-1 (Phospho-Ser307)-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target RFU values.
2.	An antibody against the nonphosphorylated counterpart of IRS-1 (Phospho-Ser307) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated IRS-1 can be used to normalize the RFU value for phosphorylated IRS-1.

IRS-1 (Phospho-Ser307) Fluorometric Cell-Based ELISA Kit -Information

Product Name:	IRS-1 (Phospho-Ser307) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:	FBCAB00018
Description:	The IRS-1 (Phospho-Ser307) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor IRS-1 (Phospho-Ser307) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated IRS-1 (Phospho-Ser307) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on IRS1 phosphorylation.
Dynamic Range:	> 5000 Cells
Detection Method:	Fluorometric
Storage/Stability:	4°C/6 Months
Reactivity:	Human, Mouse, Rat
Assay Type:	Cell-Based ELISA
Database Links:	Gene ID: 3667, UniProt ID: P35568, OMIM #: 125853/147545, Unigene #: Hs.471508
Format:	Two 96-Well Plates
NCBI Gene Symbol:	IRS1
Sub Type:	Phospho
Target Name:	Phospho-IRS-1 (Ser307)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components	Quantity
96-Well Black Cell CultureClear-Bottom Microplate	2 plates
10X TBS	24 ml
Quenching Buffer	24 ml
Blocking Buffer	50 ml
15X Wash Buffer	50 ml
Primary Antibody Diluent	12 ml
100x Anti-Phospho Target Antibody	60 µl
100x Anti-Target Antibody	60 µl
Anti-GAPDH Antibody	110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody	6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody	6 ml
Adhesive Plate Seals	2 seals

Additional equipment and materials required

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
Micropipettes capable of measuring volumes from 1 µl to 1 ml
Deionized or sterile water (ddH2O)
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

1.	Seed 200 µl of desired cell concentration in culture medium into each well of the 96-well plates. For suspension cells and loosely attached cells, coat the plates with 100 µl of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.	Incubate the cells for overnight at 37°C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µl of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µl of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µl 1x Wash Buffer for 3 minutes. The plate can be stored at 4°C for a week.
7.	Add 100 µl of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 3 minutes each time.
9.	Dispense 200 µl of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
11.	Add 50 µl of Primary Antibody Mixture P to corresponding wells for IRS-1 (Phospho-Ser307) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total IRS-1 detection. Cover the plate with parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
13.	Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.	Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.	Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.	Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

IRS-1 (Phospho-Ser307) - Protein Information

UniProt Protein Function:	IRS1: an adaptor protein that is one of the major substrates of the insulin receptor kinase. Contains multiple tyrosine phosphorylation motifs that serve as docking sites for SH2-domain-containing proteins including phosphatidylinositol 3-kinase p85 subunit and GRB-2. Phosphorylated on ser/thr residues by kinases including JNK and IKK.
UniProt Protein Details:	Protein type:Adaptor/scaffold; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 2q36 Cellular Component: intracellular membrane-bound organelle; cytoplasm; plasma membrane; caveola; cytosol; nucleus Molecular Function:insulin-like growth factor receptor binding; protein binding; signal transducer activity; protein kinase C binding; phosphoinositide 3-kinase binding; SH2 domain binding; transmembrane receptor protein tyrosine kinase adaptor protein activity; insulin receptor binding Biological Process: response to peptide hormone stimulus; phosphoinositide 3-kinase cascade; nerve growth factor receptor signaling pathway; positive regulation of glycogen biosynthetic process; negative regulation of insulin receptor signaling pathway; glucose homeostasis; signal transduction; negative regulation of insulin secretion; response to insulin stimulus; positive regulation of glucose import; mammary gland development; positive regulation of mesenchymal cell proliferation; positive regulation of cell proliferation; protein kinase B signaling cascade; epidermal growth factor receptor signaling pathway; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; positive regulation of phosphoinositide 3-kinase activity; positive regulation of fatty acid beta-oxidation; cellular response to insulin stimulus; insulin-like growth factor receptor signaling pathway; regulation of gene expression; insulin receptor signaling pathway; innate immune response; positive regulation of insulin receptor signaling pathway; lipid catabolic process; positive regulation of cell migration Disease: Diabetes Mellitus, Noninsulin-dependent
NCBI Summary:	This gene encodes a protein which is phosphorylated by insulin receptor tyrosine kinase. Mutations in this gene are associated with type II diabetes and susceptibility to insulin resistance. [provided by RefSeq, Nov 2009]
UniProt Code:	P35568
NCBI GenInfo Identifier:	547738
NCBI Gene ID:	3667
NCBI Accession:	P35568.1
UniProt Related Accession:	P35568
Molecular Weight:
NCBI Full Name:	Insulin receptor substrate 1
NCBI Synonym Full Names:	insulin receptor substrate 1
NCBI Official Symbol:	IRS1
NCBI Official Synonym Symbols:	HIRS-1
NCBI Protein Information:	insulin receptor substrate 1; IRS-1
UniProt Protein Name:	Insulin receptor substrate 1
Protein Family:	Isoleucine--tRNA ligase
UniProt Gene Name:	IRS1
UniProt Entry Name:	IRS1_HUMAN