Mouse Sonic Hedgehog/Shh N-Terminus Quantikine ELISA Kit Summary

Product Summary

The Quantikine Mouse Sonic Hedgehog N-Terminus Immunoassay is a 4.5 hour solid phase ELISA designed to measure Shh-N in cell culture supernates, serum, and plasma. It contains recombinant mouse Shh-N and antibodies raised against the recombinant factor. This immunoassay has been shown to quantitate the recombinant factor accurately. Results obtained using natural mouse Shh-N showed dose-response curves that were parallel to the standard curves obtained using the recombinant Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural mouse Shh-N.

Preparation and Storage

Background: Sonic Hedgehog/Shh

Shh (Sonic Hedgehog) is expressed in embryonic tissues that are critical for the patterning of the developing central nervous system, somite, and limb. It is also involved in whisker, hair, foregut, tooth, and bone development. Shh regulates neural and hematopoietic stem cell fate and is important for thymocyte differentiation and proliferation. In adult tissue Shh is associated with cancer development and tissue remodeling following injury. Palmitoylation and cholesterol addition of Shh contribute to its membrane tethering and assembly into multimers. Lipid modification and multimerization greatly increase Shh signaling potency. Shh can be released from the plasma membrane by the cooperative action of DISP1, SCUBE2, and TACE/ADAM17. Canonical signaling of Shh is mediated by a receptor complex that includes Patched (PTCH1, PTCH2) and Smoothened (SMO).

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.