RayBio® Phospho-AKT(Ser473) Quantitative ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in Human cell lysates. By determining phosphorylated AKT 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 quantitative measurement of Human phospho-AKT. An anti-pan AKT antibody has been coated onto a 96-well plate. Standard and Samples are pipetted into the wells and AKT present in a sample is bound to the wells by the immobilized antibody. The wells are washed and rabbit anti-phospho-AKT(Ser473) antibody is used to detect phosphorylated AKT. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG 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 AKT(Ser473) bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450nm.
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
Distilled or deionized water
100 ml and 1 liter graduated cylinders
Tubes to prepare sample dilutions
Protease and Phosphatase inhibitors
Precision pipettes to deliver 2 µl to 1 ml volumes
Adjustable 1-25 ml pipettes for reagent preparation
Benchtop rocker or shaker
Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
Prepare all reagents and samples as instructed in the manual.
Add 100 µl of sample or positive control to each well.
Incubate 2.5 h at RT or O/N at 4 °C.
Add 100 µl of prepared primary antibody to each well.
Incubate 1 h at RT.
Add 100 µl of prepared 1X HRP-Streptavidin to each well.
Incubate 1 h at RT.
Add 100 µl of TMB One-Step Substrate Reagent to each well.
Incubate 30 min at RT.
Add 50 µl of Stop Solution to each well.
Read at 450 nm immediately.
Typical Data
Positive Control
PDGFBB Stimulation of NIH3T3 Cell Lines
NIH3T3 cells were untreated or treated with PDGFBB for 10 min. Cell lysates were analyzed using this phosphoELISA kit and western blot.
Storage/Stability
Upon receipt, the kit should be stored at -20 °C. Please use within 6 months from the date of shipment. After initial use, Wash Buffer Concentrate (Item B), Assay Diluent (Item E), TMB One-Step Substrate Reagent (Item H), HRP-Streptavidin (Item G), Stop Solution (Item I) and Cell Lysate Buffer (Item J) should be stored at 4 °C to avoid repeated freeze-thaw cycles. Return unused wells to the pouch containing desiccant pack, reseal along entire edge and store at -20 °C. Reconstituted Positive Control (Item K) should be stored at -70 °C.
