RayBio® Phospho-AKT/GSKß/mTor ELISA Kit. This assay semi-quantitatively measures phosphorylated AKT/GSK-beta/mTor in lysate samples.

Antigen Information

Accession Number:

P31749

Gene ID:

Gene Symbols:

AKT1, PKB, RAC

Protein Name & Synonyms:

RAC-alpha serine/threonine-protein kinase (EC 2.7.11.1) (Protein kinase B) (PKB) (Protein kinase B alpha) (PKB alpha) (Proto-oncogene c-Akt) (RAC-PK-alpha)

Assay Format

Pathway:

Insulin Signaling
mTOR Signaling
PI3K-AKT Signaling

Specificity:

The antibody pair provided in this kit recognizes phosphorylated AKT (S473), GSK3β (S9), mTOR (S2448), and total AKT, GSK3β, mTOR in Human and Mouse Cell Lysates.

Number of Targets Detected:

Species Detected:

Human
Mouse

Compatible Sample Types:

Cell Lysates
Tissue Lysates

Design Principle:

Sandwich-based

Method of Detection:

Colorimetric

Quantitative/Semi-Quantitative:

Semi-Quantitative

Solid Support:

96-well Microplate

Product Specifications

Size:

1, 2, or 5 x 96-Well Strip Microplate Kit

Introduction

RayBio® AKT Pathway ELISA Sample 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 AKT, GSK3β and mTOR 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-AKT, total AKT, phospho-GSK3β, total GSK3β, phospho-mTOR and total mTOR. For each target, a capture antibody has been coated onto microwells. Samples are pipetted into the wells and target protein present in a sample is bound to the wells by the immobilized antibody. The wells are washed and a detection antibody is used to detect the captured target protein. After washing away unbound antibody, an HRP-conjugated secondary antibody is pipetted to 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 target protein bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Product Features

Simultaneously measure Phosphorylated protein and pan protein in one experiment (for normalization purpose)
Screen numerous different cell lysates without performing a Western Blot analysis
Minimal hands-on time, convenient, and non-radioactive material

Target Name/s

Phosphorylated: AKT (S473), GSK3β (S9), mTOR (S2448)
Total: AKT, GSK3β and mTOR

Application Notes

Kit Components

1 Microplate (Item A)
Wash Buffer Concentrate (20x) (Item B)
Assay Diluent (Item E)
Detection Antibody Akt (Ser473) (Item C-1)
Detection Antibody Akt (Item C-2)
Detection Antibody GSK3β (S9) (Item C-3)
Detection Antibody GSK3β (Item C-4)
Detection Antibody mTOR (S2448) (Item C-5)
Detection Antibody mTOR (Item C-6)
HRP-conjugated Anti-rabbit IgG (Item D-1)
HRP-conjugated Streptavidin (Item D-2)
TMB One-Step Substrate Reagent (Item H)
Stop Solution (Item I)
Cell Lysate Buffer (Item J)
Positive Control 3T3S001-1 (Item K)

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.

Microplate Layout

Phospho-AKT/GSKß/mTor ELISA(图1)

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), 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. Item D, store at 2-8°C for up to one month (store at -20°C for up to 6 months, avoid repeated freeze-thaw cycles). Reconstituted Positive Control (Item K) should be stored at -70°C.