The RayBio® Phospho-IRS1 (Ser318) and Total IRS1 ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in human and mouse cell lysates.
RayBio® Phospho-IRS1 (Ser318) and Total IRS1 ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in Human and Mouse cell lysates. By determining phosphorylated IRS1 protein in your experimental model system, you can verify pathway activation in your cell lysates. You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blotting analysis.
This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of Human and Mouse phospho-IRS1 and total IRS1. An anti-pan IRS1 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and IRS1 present in a sample is bound to the wells by the immobilized antibody and the wells are washed. In select wells, rabbit anti-phospho-IRS1 (Ser318) antibody is added to detect phosphorylated IRS1. In the remaining wells, biotinylated anti-pan-IRS1 antibody is used to detect pan IRS1. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG or HRP-Streptavidin is pipetted into the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of IRS1 (Ser318) or pan IRS1 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
Product Features
Rapidly measure phosphorylated protein in lysates
Screen numerous different cell lysates without performing a Western Blot analysis
Minimal hands-on time, convenient, and non-radioactive material
Application Notes
Kit Components
Pre-Coated 96-well Strip Microplate
Wash Buffer
Anti-Phospho Antibody
HRP-Conjugated Secondary Antibody
Assay Diluent
TMB One-Step Substrate
Stop Solution
Lysis Buffer
Positive Control Sample
Other Materials Required
Microplate reader capable of measuring absorbance at 450 nm
Protease and Phosphatase inhibitors
Benchtop rocker or shaker
Precision pipettes to deliver 2 µl to 1 ml volumes
Adjustable 1-25 ml pipettes for reagent preparation
100 ml and 1 liter graduated cylinders
Absorbent paper
Distilled or deionized water
Log-log graph paper or computer and software for ELISA data analysis
Tubes to prepare the positive control or sample dilutions
Protocol Outline
Prepare all reagents, samples and positive control as instructed.
Add 100 μl positive control or sample to each well. Incubate 2.5 hours at room temperature or overnight at 4°C with gentle shaking.
Add 100 μl prepared detection antibody to each well. Incubate for 1 hour at room temperature with gentle shaking.
Add 100 μl prepared HRP-Conjugated solution. Incubate for 1 hour at room temperature with gentle shaking.
Add 100 μl TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature.
Add 50 μl Stop Solution to each well. Read at 450 nm immediately.
Typical Data
Positive Control
HeLa cells were treated with TNFa and Calyculin A. Cells were solubilzed at 4 x 107 cells/ml in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA.
Insulin Stimulation of MCF-7 Cell Lines
MCF-7 cells were untreated or treated with Insulin. Cell lysates were analyzed using this phosphoELISA and Western Blot.
Storage/Stability
The entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles. For extended storage, it is recommended to store at -80°C.
