Human Tie-2 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human Tie-2 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human Tie-2 in cell culture supernates, serum, and plasma. It contains NS0-expressed recombinant human Tie-2 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human Tie-2 showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural human Tie-2.

Preparation and Storage

Background: Tie-2

Tie-2, also known as Tek, is a transmembrane receptor tyrosine kinase (RTK) that functions as a receptor for Angiopoietin family proteins. It is expressed by embryonic and adult endothelial cells, hematopoietic stem cells, and a circulating population of proangiogenic monocytes. Tie-2 signaling is activated by Angiopoietins-1 and -4, while it can be either activated or inhibited by Angiopoietins-2 and -3. Tie-2 plays an important role in maintaining vascular integrity by mediating endothelial cell-smooth muscle cell communication and inhibiting endothelial cell apoptosis. It is also required for embryonic development of the endocardium. In addition, Tie-2 signaling mediates the quiescence of bone marrow stem cells in response to osteoblast-produced Angiopoietin-1. This quiescence is critical for maintaining an ongoing hematopoietic capability in the bone marrow.

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.