Human Tie-1 DuoSet ELISA Summary

Product Summary

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human Tie-1. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.

Preparation and Storage

Background: Tie-1

Tie-1/Tie and Tie-2/Tek are receptor tyrosine kinases with unique structural characteristics including two immunoglobulin-like domains flanking three epidermal growth factor (EGF)-like domains, followed by three fibronectin type III-like repeats in the extracellular region, and a split tyrosine kinase domain in the cytoplasmic region. Tie-2 is a receptor for the angiopoietin (ANG) family: ANG-1, ANG-2, and ANG-3 (mouse)/-4 (human). It is involved in vascular stabilization and remodeling. Although less well understood, Tie-1 may also act as an ANG receptor, possibly in complex with Tie-2.

Human Tie-1 cDNA encodes a 1138 amino acid (aa) residue precursor protein with a 24 residue putative signal peptide, a 735 residue extracellular domain and a 354 residue cytoplasmic domain. Ligands which bind and activate Tie-1 have not been identified. Based on gene targeting studies, the in vivo functions of Tie-1 have been shown to be related to endothelial cell differentiation and the maintenance of integrity of the endothelium.

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.