Transforming Human Growth Factor Beta Receptor I/II (TGFβR)
| Product Family | Product Number | Product Description | Technical Manual |
| IB1200 TGFβR |
IB12001-32 | Human TGFβR Reporter Assay System, 3 x 32 assays in 96-well format | Technical Manual |
| IB12001 | Human TGFβR Reporter Assay System, 1 x 96-well format assays | Technical Manual | |
| IB12002 | Human TGFβR Reporter Assay System, 1 x 384-well format assays | Technical Manual |
Transforming Human Growth Factor Beta Assay Kit
This Human Transforming Growth Factor beta Receptor (TGFβ) assay kit is an all-inclusive firefly luciferase reporter assay system that includes in addition the the TGFβ Reporter Cells, two optimized media for use in recovering the cryopreserved cells and for diluting test samples. Also included is the reference agonist TGF-b1, Luciferase Detection Reagents, and a cell culture-ready assay plate.
TGFβ Reporter Cells are prepared using INDIGO’s proprietary CryoMite™ process. This cryo-preservation method yields high cell viability post-thaw, and provides the convenience of immediately dispensing healthy, division-competent reporter cells into assay plates. There is no need for intermediate spin-and-wash steps, viability determinations, or cell titer adjustments.
INDIGO’s TGF beta assay kits feature a luciferase detection reagent specially formulated to provide stable light emission between 5 and 90+ minutes after initiating the luciferase reaction. Incorporating a 5-minute reaction-rest period ensures that light emission profiles attain maximal stability, thereby allowing assay plates to be processed in batch. By doing so, the signal output from all sample wells, from one plate to the next, may be directly compared within an experimental set.
TGF beta assay kits are offered in different assay formats to accommodate researchers' needs: 3x 32, 1x 96, and 1x 384 assay formats for screening small numbers of test compounds, as well as custom bulk reagents for HTS applications.
Bulk assay reagents can be custom manufactured to accommodate any scale of HTS. Please inquire.
Transforming Human Growth Factor Beta Assay Services
The primary application of INDIGO's cell-based nuclear receptor assays are to quantitatively assess the bioactivity of a test compound as an agonist (activator) or antagonist (inhibition of an agonist response) of a given receptor. Service assays include a positive control reference compound and 'vehicle' control for every experiment. A formal study report and all data files are provided to the client upon completion of the study. To receive a quote for your proposed study, complete & submit the online "Request a Quote" form or contact an INDIGO Customer Service Representative to discuss your desired study parameters.
Transforming Human Growth Factor Beta Background
The TGFβ assay uses proprietary human cells that provide constitutive expression of the Human type I and type II Transforming Growth Factor beta Receptors (TGFβ I/II), both of which are transmembrane serine/threonine kinase receptors. Receptor activation results when TGFβ binds to RII, which then phosphorylates and forms a heterodimer complex with RI. The mechanisms of TGFβ-RI/RII activation and ensuing signal transduction cascade have been well studied and involve the phosphorylation and interplay of a variety of Smad regulatory proteins and transcription factors. Additionally, cross-talk between the activated TGFβ and NF-kB signal transduction pathways have been characterized.
INDIGO's Reporter Cells include the luciferase reporter gene functionally linked to tandem TGFβ Response Sequences (TRS) derived from the human plasminogen activator inhibitor-1 (PAI-1) promoter, a well-characterized TGFβ responsive target gene. The TRS sequences are readily bound by activated dimeric Smad3/Smad4 to initiate formation of a transcription complex. This binding leads to the activation of downstream substrates and regulatory proteins, including transcription of different target genes that function in differentiation, chemotaxis, proliferation, and activation of many immune cells. Among TGFβR's functions is the regulation of inflammatory processes, particularly in the gut. It also plays a crucial role in stem cell differentiation, as well as T-cell regulation and differentiation.
Quantifying changes in luciferase activity in drug-treated cells versus untreated (or vehicle-treated) reporter cells provides a sensitive surrogate measure of drug-induced changes in TGFβR activity. The principal application of this TGFβ receptor assay is in the screening of compounds / drug candidates to quantify any functional activity, either agonist or antagonist, that they may exert against the human TGFβ receptor.
Transforming Human Growth Factor Beta Assay Data
Agonist dose-response of the TGFβR. Dose-response analyses of TGFβR Reporter Cells were performed according to the protocol provided in this Technical Manual. TGFβR Reporter Cells were treated with TGF-β using a range of 7 concentrations (n = 4/conc.) generated in 3-fold decrements: 3.0, 1.0, .33, .11, .037, .012 and 0.0041 ng/mL, and including ‘untreated’ (i.e., ‘vehicle only’) control wells, as described in Appendix 1 of the assay technical manual. Luminescence/well was quantified and the average relative light units (RLU) and corresponding standard deviation (SD), percent coefficient of variation (%CV) and Fold-Activation values were determined for each treatment concentration. Z’ values were calculated as described by Zhang, et al. (1999). Non-linear regression analyses and IC50 calculations were performed using GraphPad Prism software. These data confirm the robust performance of this TGFβR Assay and demonstrate its suitability for use in HTS applications.
Agonist dose-response of the TGFβR. Dose-response analyses of TGFβR Reporter Cells were performed according to the protocol provided in this Technical Manual. TGFβR Reporter Cells were treated with TGF-β using a range of 7 concentrations (n = 4/conc.) generated in 3-fold decrements: 3.0, 1.0, .33, .11, .037, .012 and 0.0041 ng/mL, and including ‘untreated’ (i.e., ‘vehicle only’) control wells, as described in Appendix 1 of the assay technical manual. Luminescence/well was quantified and the average relative light units (RLU) and corresponding standard deviation (SD), percent coefficient of variation (%CV) and Fold-Activation values were determined for each treatment concentration. Z’ values were calculated as described by Zhang, et al. (1999). Non-linear regression analyses and IC50 calculations were performed using GraphPad Prism software. These data confirm the robust performance of this TGFβR Assay and demonstrate its suitability for use in HTS applications.