AKT Pathway (AKT/GSK/p70 S6K) Activation Profile Multispecies InstantOne™ ELISA Kit

Catalog number: 85-86048-11

Invitrogen™  Related applications: Protein Assays and Analysis

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Description

The InstantOne™ ELISA is specifically engineered for accurate measurement of phosphorylated AKT 1/2/3 (Ser473) (human, mouse, hamster, rat), phosphorylated GSK3beta (Ser9) (human, mouse, rat) and phosphorylated p70S6K (Thr389) (human, mouse) in cell lysates. The InstantOne™ ELISA kit allows for fast analysis of samples in approximately one hour. All reagents used in a traditional sandwich ELISA are added in solution to a plate followed by a wash step and detection with the TMB colorimetric substrate.

AKT, which exists as multiple isoforms, is one of the principle kinases activated by phosphoinositide 3-kinase (PI3K). Activation of PI3K results in the generation of phosphatidylinositol (3,4,5)-triphosphate (PIP3). These lipid second messengers bind to the pleckstrin homology domain of AKT to promote its translocation to the plasma membrane for activation via phosphorylation at Thr308 and Ser473 by PDK1 and the mTOR TORC2 complex, respectively. Phosphorylation at both these sites is required for full activation of AKT Ser/Thr kinase activity. AKT phosphorylates over 50 known substrates, including GSK3b, p70S6K, AS160, PRAS40, TSC 1, TCS 2, Raf-1, Bad, the FOXO family of transcription factors, and PFK2. Due to its established critical role in numerous biological processes, AKT is a frequent target for oncology-related drug development.

Glycogen Synthase Kinase 3 (GSK3) is a Ser/Thr kinase that exists as two isoforms, GSK3a and GSK3ß. Unlike other kinases, phosphorylation at Ser9 and Ser21 for GSK3alpha and GSK3beta, respectively, leads to inactivation of this enzyme. GSK3 has been implicated in several diseases, including cancer and diabetes, through its phosphorylation of glycogen synthase. GSK3 has also been associated with neurodegenerative diseases such as Alzheimer's disease due to its phosphorylation of the neuronal Tau protein.

p70 S6 Kinase (p70S6K) is a member of the ribosomal S6 kinase family of Ser/Thr kinases. P70 S6K activity is controlled by multiple signaling pathways, including the MAPK, phosphoinositide-3 kinase (PI3K), and mTOR pathways. This regulation results in the phosphorylation of multiple sites of p70 S6K, including Thr389 within the linker domain, which is required for full activation. Activated p70 S6K phosphorylates several residues on the S6 ribosomal protein, thereby leading to increased protein synthesis.

Suitable Sample Types
Tested on cell culture samples. Expected to work on many other sample types, but have not been tested at this time.

Reported Application
ELISA

For Research Use Only. Not for use in diagnostic procedures.

Specifications

Assay Range: See product documentation
Assay Sensitivity: See product documentation
Detection Method: Colorimetric
For Use With (Equipment): Microplate Reader
Former Identifiers: eBioscience
Gene Aliases: AKT, CWS6, PKB, PKB-ALPHA, PRKBA, RAC, RAC-ALPHA, GSK3B, PS6K, S6K, S6K-beta-1, S6K1, STK14A, p70 S6KA, p70(S6K)-alpha, p70-S6K, p70-alpha
Gene ID: 207, 2932, 6198
Gene Name: AKT serine/threonine kinase 1, glycogen synthase kinase 3 beta, ribosomal protein S6 kinase B1
Gene Symbol: AKT1, GSK3B, RPS6KB1
Immunoassay Kit Format: Coated ELISA Kit
Label or Dye: HRP (Horseradish Peroxidase)
Product Line: InstantOne™
Product Size: 96 tests
Protein Name: Glycogen synthase kinase-3 beta, RAC-alpha serine/threonine-protein kinase, Ribosomal protein S6 kinase beta-1
Sample Type (Specific): Cell Extracts
Target (Kit Named): AKT Pathway (AKT/GSK/p70 S6K) Activation Profile
Target Species (Named): Multispecies
Target Species (Validated): Human, Mouse
UniProt ID: P23443, P31749, P49841

Contents & storage

The kit includes sufficient reagents to perform 32 wells of each of the three specific targets for a total of 96 wells.
User Manual
InstantOne™ Assay Plate (Part No. IOAP96) - 96 well (sealed)
Cell Lysis Buffer (5X) (Part No. IOCLB1) - 8 mL
Enhancer Solution (Part No. IOES1) - 0.5 mL
Wash Buffer (10X) (Part No. IOWB1) - 8 mL
phospho-GSK3b (Ser9) Capture Antibody Reagent (Part No. IOPGSKCAR1T) - 1 x 1.0 mL
phospho-AKT 1/2/3 (Ser473) Capture Antibody Reagent (Part No. IOPAKTACAR1T) - 1 x 1.0 mL
phospho-p70S6K (Thr389) Capture Antibody Reagent (Part No. IOPS6KCAR1T) - 1 x 1.0 mL
phospho-GSK3b (Ser9) Detection Antibody Reagent (Part No. IOPGSKDAR1T) - 1 x 1.0 mL
phospho-AKT 1/2/3 (Ser473) Detection Antibody Reagent (Part No. IOPAKTADAR1T) - 1 x 1.0 mL
phospho-p70S6K (Thr389) Detection Antibody Reagent (Part No. IOPS6KDAR1T) - 1 x 1.0 mL
Positive Control Cell Lysate (lyophilized) (Part No. IOMGAPPC1) - 1 ea
Detection Reagent (TMB Substrate) (Part No. IODR1) - 10 mL
Stop Solution (Part No. IOSS1) - 10 mL
Plate Seal - 1 ea

Documents

Manuals & protocols