Human TGF-alpha Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human TGF-alpha Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human TGF-alpha in cell culture supernates, serum, plasma, and human milk. It contains E. coli-expressed recombinant human TGF-alpha and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human TGF-alpha 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 naturally occurring human TGF-alpha.

Preparation and Storage

Background: TGF-alpha

TGF-alpha is a member of the EGF family of cytokines that are synthesized as transmembrane precursors with soluble forms released by proteolytic cleavage. Membrane-bound proTGF-alpha is biologically active and plays a role in mediation of cell-cell adhesion and in juxtacrine stimulation of adjacent cells. Expression of TGF-alpha is widespread in tumors and transformed cells. TGF-alpha is also expressed in normal tissues during embryogenesis and in adult tissues, including pituitary, brain, keratinocytes and macrophages. TGF-alpha binds and activates the EGF receptor. It induces fibroblast proliferation, epithelial development, keratinocyte migration, and angiogenesis.

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.