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
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 |
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