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Human Phospho-AKT (S473) Quantitative ELISA

RayBio® Human Phospho-AKT (S473) Quantitative ELISA Kit. This assay quantitatively measures phosphorylated AKT (Ser473) in lysate samples.

$496.00  
PEL-AKT-S473-Q-1
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Antigen Information

Accession Number:
  • P31749
Gene ID:
  • 391
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 human Phospho-AKT (Ser473).
Number of Targets Detected:
1
Species Detected:
  • Human
Compatible Sample Types:
  • Cell Lysates
  • Tissue Lysates
Design Principle:
  • Sandwich-based
Method of Detection:
Colorimetric
Quantitative/Semi-Quantitative:
Quantitative
Solid Support:
96-well Microplate

Product Specifications

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

Introduction

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
  1. Prepare all reagents and samples as instructed in the manual.
  2. Add 100 µl of sample or positive control to each well.
  3. Incubate 2.5 h at RT or O/N at 4 °C.
  4. Add 100 µl of prepared primary antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 µl of prepared 1X HRP-Streptavidin to each well.
  7. Incubate 1 h at RT.
  8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl of Stop Solution to each well.
  11. Read at 450 nm immediately.

Typical Data

Positive Control
AKT (S473) - 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.
AKT (S473) - ELISA Kit
AKT (S473) - 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.

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Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.