Human Dkk-1 Quantikine ELISA Kit Summary

Product Summary

The Quantikine^® Human Dkk-1 immunoassay is a 4.5 hour solid phase ELISA designed to measure human Dkk-1 in cell culture supernates, serum-free cell culture supernates, serum, and platelet-poor plasma. It contains Sf 21-expressed recombinant human Dkk-1 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human Dkk-1 showed dose-response 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 of natural human Dkk-1.

Preparation and Storage

Background: Dkk-1

Members of the dickkopf-related protein family (Dkk-1, -2, -3, and -4) are secreted proteins with two cysteine-rich domains separated by a linker region. Dkk-3 and -4 also have one prokineticin domain. Dkk-1, -2, and -4 function as antagonists of canonical Wnt signaling by binding to LRP5/6, preventing interaction of LRP5/6 with Wnt-Frizzled complexes. Dkk-1, -2, and -4 also bind cell surface Kremen-1 or -2 and promote the internalization of LRP5/6. Antagonistic activity of Dkk-3 has not been demonstrated. Dkk proteins have distinct patterns of expression in adult and embryonic tissues and have a wide range of effects on tissue development and morphogenesis. The Dkk family also includes Soggy, which is homologous to Dkk-3 but not to the other family members. Soggy has not been shown to inhibit Wnt signaling, and its role in the pathway is unclear.

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.