Human/Mouse/Rat Total Akt1 DuoSet IC ELISA Summary

Product Summary

This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure total Akt1 in cell lysates. An immobilized capture antibody specific for Akt1 binds both phosphorylated and unphosphorylated Akt1. After washing away unbound material, a biotinylated detection antibody is used to detect both phosphorylated and unphosphorylated protein, utilizing a standard Streptavidin-HRP format.

Preparation and Storage

Background: Akt1

Akt1, Akt2, and Akt3 make up a family of serine/threonine kinases, often activated downstream of growth factor receptors and PI 3-Kinase. Each Akt family member contains an N-terminal pleckstrin homology (PH) domain, a central kinase domain, and a C-terminal regulatory domain. They have putative roles in cell proliferation and survival, and Akt perturbations are thought to play an important role in tumorigenesis.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1、Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2、Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3、Add 50 μL of Assay Diluent to each well.

4、Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5、Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6、Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7、Aspirate and wash 5 times.

8、Add 100 μL Substrate Solution to each well.

9、Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.