Overview of Transfluor Assay
By attaching a fluorescent label to beta-arrestin, the location of the receptor-arrestin complex may be monitored during receptor activation. Since desensitization only occurs with an activated receptor, monitoring beta-arrestin translocation and subsequent receptor recycling provides a method to detect the activation of any GPCR.
Studying this mechanism enables researchers to identify the impact of GPCR ligands on a variety of physiological processes including behavior, mood, inflammation, nervous system, and olfactory responses. In addition, researchers can gain insight into the complexities of receptor degradation and recycling for a given target.
- Validated with over 100 GPCRs, the Transfluor assay provides the assurance to work across all GPCR classes(Class I, II, III), regardless of interacting G-protein (Gs, Gi/o and Gq/11).
- Single read-out is compatible with all GPCR subtypes, including Gi, Gs, Go, Gq, eliminating the need for multiple GPCR assays.
- No GPCR labeling or tagging avoids custom molecular biology that may alter your target's response.
- Universal format requires no prior knowledge of interacting G-protein, enabling orphan GPCR screening.
- GFP-based technology does not require additional substrates to monitor beta-arrestin translocation.
- MetaXpress® Application Module automates analysis of beta-arrestin movement to the cell membrane (pits) and endocytic vesicles.
- Ligand Independent Translocation (LITe™) Assay is an agonist-independent assay used to verify the translocation of beta-arrestin-GFP in orphan GPCRs.
Data of Transfluor Assay
High Content Images of Transfluor Assay
ß2AR-expressing cells were stimulated with isoproterenol. Left: control, center: pits, right: vesicles. Top: Transfluor Assay imaged with ImageXpress. Bottom: Transfluor Assay imaged with Discovery-1.
Dose Response to Isoproterenol
Segmentation and analysis run with the GPCR Cycling Application Module for MetaXpress® High-Content Image Acquisition and Analysis Software. Error bars show standard error. Top: pits, bottom: vesicles.
Partial GPCR Listing
The Transfluor technology monitors receptor activity by detecting movement of beta-arrestin-GFP in the cell. A partial listing of GPCRs that have been shown to translocate beta-arrestin-GFP is shown below.
Other Receptor Listing
- 12 Drosophila GPCRs
- Fz4 frizzled receptor
- TßRIII transforming growth factor-ß
Technology of Transfluor Assay
Orphan GPCR Assay
In contrast to current methods of screening GPCRs, the Transfluor technology is based on the mechanism for termination of GPCR signaling, known as receptor desensitization. This mechanism is shared by virtually all GPCRs and is activated by ligand binding. Transfluor technology requires no prior knowledge of the interacting G-protein. This important feature of the Transfluor technology makes it ideal for screening orphan GPCRs (oGPCR).
A proprietary technique to assist in validating orphan GPCR screens, called LITe™ (Ligand Independent Translocation), is an agonist-independent assay used to verify the translocation of ß arrestin-GFP in orphan GPCRs.
Resources of Transfluor Assay
The ImageXpress® Micro Confocal/ XLS Widefield High-Content Imaging System from Molecular Devices includes optional Fluidics and Environmental Control modules, enabling single-channel…
|Product Name||Part Number|
|Transfluor® Evaluation Kit (Non Commercial Use)||R8177|
|Transfluor® Evaluation Kit (Commercial Use)||R8178|
|U2OS (G418) rat b-arrestin 2-RrGFP parental cell lines||TF-U2OS-rBA2GFP|
Transfluor Evaluation Kit
The Transfluor Evaluation Kit enables use of the technology for a limited, one-time, six month evaluation period. The kit provides all the necessary tools for evaluating the technology with the users' own instrumentation and GPCRs. This allows users to become comfortable with the technology, before proceeding to a tiered licensing option that meets users' specific needs.
Transfluor Evaluation Kit Components
- U-2 OS β-arrestin2-GFP cells (Parental cell line, to assist with determination of the Transfluor response to end-users' choice of GPCR)
- Transfluor Demo Plate (Imaging grade plate with fixed cells to assist with instrument validation and image acquisition and analysis)
- Limited Label License
- Transfluor Evaluation Kit Product Insert
- U-2 OS β-arrestin2-GFP cells Product Insert
- Transfluor Quick Start Protocol
Transfluor User Guide (complete with protocols and troubleshooting guidelines)
Please contact your local sales representative for detailed information regarding this kit.
Tiered Licensing Options
Use of the Transfluor technology requires the purchase of a Transfluor Technology Use License from Molecular Devices which is renewable on an annual basis. A scalable license structure is available from research up to high-throughput screening including the following examples:
- Transfluor Assay Research Site License
- Transfluor Assay Research Global License
- Transfluor Screening Site License
- Transfluor Assay Research and Screening Site License
Two additional third-party licenses are required to run the Transfluor assay: a GFP license (Molecular Devices recommends the Prolume Renilla reniformis (Rr) GFP license) and BioImage's fluorescence redistribution license. Terms are as follows:
- Prolume license is renewable on an annual basis for a nominal fee.
- BioImage license is a one-time fee.
- Prolume and BioImage licenses both provide worldwide coverage.
To streamline your process, you can obtain these licenses directly from Molecular Devices. Alternatively, you can also obtain them directly from Prolume and BioImage.
Please contact your local sales representative for detailed information regarding these licenses. In conjunction with the Transfluor license, Molecular Devices also offers a stably transfected GFP beta-arrestin starter cell line and relevant plasmids.
Compatible Products & Services of Transfluor Assay
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