Mouse/Rat/Porcine/Canine TGF-beta 1 Quantikine ELISA Kit Summary

Product Summary

Transforming Growth Factor Beta 1, 2, and 3 (TGF-beta 1, TGF-beta 2, and TGF-beta 3) are highly pleiotropic cytokines that virtually all cell types secrete. TGF-beta molecules are proposed to act as cellular switches that regulate processes such as immune function, proliferation, and epithelial-mesenchymal transition. Targeted deletions of these genes in mice show that each TGF-beta isoform has some non-redundant functions: TGF-beta 1 is involved in hematopoiesis and endothelial differentiation; TGF-beta 2 affects development of cardiac, lung, craniofacial, limb, eye, ear, and urogenital systems; and TGF-beta 3 influences palatogenesis and pulmonary development. The full range of in vitro biological activities of TGF-beta 5 has not yet been explored. However, TGF-beta 1, TGF-beta 2, TGF-beta 3, and TGF-beta 5 have been found to be largely interchangeable in an inhibitory bioassay, and it is anticipated that TGF-beta 5 will show a spectrum of activities similar to the other TGF-beta family members. To date, the production of TGF-beta 5 has only been demonstrated in Xenopus.

TGF-beta ligands are initially synthesized as precursor proteins that undergo proteolytic cleavage. The mature segments form active ligand dimers via a disulfide-rich core consisting of the characteristic 'cysteine knot'. TGF-beta signaling begins with binding to a complex of the accessory receptor betaglycan (also known as TGF-beta RIII) and a type II serine/threonine kinase receptor termed TGF-beta RII. This receptor then phosphorylates and activates a type I serine/threonine kinase receptor, either ALK-1 or TGF-beta RI (also called ALK-5). The activated type I receptor phosphorylates and activates Smad proteins that regulate transcription. Use of other signaling pathways that are Smad-independent allows for distinct actions observed in response to TGF-beta in different contexts.

Preparation and Storage

Background: TGF-beta 1

The IL-17 family is comprised of at least six proinflammatory cytokines that share a conserved cysteine-knot structure but diverge at the N-terminus. IL-17 family members are glycoproteins secreted as dimers that induce local cytokine production and recruit granulocytes to sites of inflammation. IL-17 is induced by IL-15 and IL-23, mainly in activated CD4+ T cells distinct from Th1 or Th2 cells. IL-17F is the most homologous to IL-17, but is induced only by IL-23 in activated monocytes. IL-17B binds the IL-17B receptor, but not the IL-17 receptor; it is most homologous with IL-17D, which is expressed by resting CD4+ T cells and CD19+ B cells. IL-17E is mainly produced by Th2 cells and recruits eosinophils to lung tissue. IL-17C has a very restricted expression pattern but has been detected in adult prostate and fetal kidney libraries.

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.