Human Phospho-EphA2 DuoSet IC ELISA Summary

Product Summary

This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure tyrosine-phosphorylated human EphA2 in cell lysates. An immobilized capture antibody specific for EphA2 binds both phosphorylated and unphosphorylated EphA2. After washing away unbound material, a biotinylated detection antibody is used to detect only phosphorylated receptor, utilizing a standard HRP format.

Preparation and Storage

Background: EphA2

Cell migration is influenced by chemodirectants including a large family of receptor tyrosine kinases collectively referred to as Ephs. Ephs are divided into two classes based on their extracellular structure and ligand specificity. Eph receptors that bind preferentially to ephrin-A ligands are referred to as EphA. All Eph receptors contain an extracellular region consisting of a globular domain, a cysteine-rich domain, and two fibronectin type III domains. This is followed by the transmembrane region and cytoplasmic region. The cytoplasmic region contains a juxtamembrane motif with two tyrosine residues, a kinase domain, and a conserved sterile alpha motif (SAM) in the carboxy tail with one conserved tyrosine residue.

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.