Phospho-AKT/GSKß/mTor ELISA

Cell Lysates
Tissue Lysates

Colorimetric

96-well Microplate

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 G
S
K3β (
S9) (
Item C-3)
Detection Antibody G
S
K3β (
Item C-4)
Detection Antibody m
T
O
R (
S2448) (
Item C-5)
Detection Antibody m
T
O
R (
Item C-6)
H
R
P-conjugated Anti-rabbit Ig
G (
Item D-1)
H
R
P-conjugated Streptavidin (
Item D-2)
T
M
B One-
Step Substrate Reagent (
Item H)
Stop Solution (
Item I)
Cell Lysate Buffer (
Item J)
Positive Control 3
T3
S001-1 (
Item K)

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 R
T or O/
N at 4 °
C.
Add 100 µl of prepared primary antibody to each well.
Incubate 1 h at R
T.
Add 100 µl of prepared 1
X H
R
P-
Streptavidin to each well.
Incubate 1 h at R
T.
Add 100 µl of T
M
B One-
Step Substrate Reagent to each well.
Incubate 30 min at R
T.
Add 50 µl of Stop Solution to each well.
Read at 450 nm immediately.

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)

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.