Human Phospho-EphB4 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 EphB4 in cell lysates. An immobilized capture antibody specific for EphB4 binds both phosphorylated and unphosphorylated EphB4. 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: EphB4

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-B ligands are referred to as EphB. All Eph receptors contain an N-terminal Ig-like domain, a cysteine-rich region with 19 conserved cysteines and two fibronectin type III domains. The cytoplasmic region contains a typical tyrosine kinase organization.

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.