The cookie settings on this website are set to 'allow all cookies' to give you the very best experience. Please click Accept Cookies to continue to use the site.

Human/Mouse/Rat Phospho-AKT (S473) and Total Akt ELISA

(No reviews yet) Write a Review
SKU:
RHHU0002

Description

  • Accession Number:P31749
  • Gene ID: 391
  • Gene Symbol: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, mouse and rat Phospho-Akt (pSer473) + pan Akt.
  • Number of Targets Detected:2
  • Species Detected:Human, Mouse, Rat
  • Compatible Sample Types: Cell & tissue lysates
  • Design Principle:Sandwich-based
  • Method of Detection:Colorimetric
  • Quantitative/Semi-Quantitative: Semi-Quantitative
  • Solid Support: 96-well Microplate
Product Specifications
  • Size:96-Well Strip Microplate Kit

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

Application Notes

Kit Components:
  • Pre-Coated 96-well Strip Microplate
  • Wash Buffer
  • Anti-Phospho Antibody
  • Anti-Pan Antibody
  • HRP-Conjugated Secondary Antibody
  • Streptavidin-Conjugated HRP
  • 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.

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.

View AllClose

Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
View AllClose