Quantcast
Industry Cortex

Document Brief

P r a c t i c a l Ap P l i c at i o n s...

A no-wash indicator for high-throughput calcium detection...

H1 histamine GPCR screening using PremoTM Cameleon calcium sensor....

Second messengers such as cAMP and Ca2+ are key components of cell signaling pathways. Monitoring these transient second messengers in their native states requires an easy-to-use, effective tool for high-throughput screening (HTS). The combination of a fluorescent protein biosensor and efficient BacMam delivery technology provides an excellent method for expressing genetically encoded sensors in mammalian cells. Using this approach, we screened a H1 histamine G protein coupled receptor (GPCR) stably expressed in HEK 293T cells and demonstrated the utility of PremoTM Cameleon Calcium Sensor for high-throughput calcium flux measurements....

A powerful combination for cell-based assays...

PremoTM Cameleon Calcium Sensor is based on the CFP YFP fluorescence resonance energy transfer (FRET) pair linked by a calcium-responsive calmodulin-M13 unit.2 The gene construct is carried on a baculovirus chromosome, which efficiently delivers its genetic payload to mammalian cells. Because only the transgene cassette with a mammalian promoter (here CMV) is recognized by the mammalian cell machinery, only the PremoTM Cameleon Calcium Sensor is expressed (Figure 1)....

The BacMam advantage...

Promoter CFP Calmodulin- M13 YFP (Venus)...

BacMam-mediated transduction is efficient and reproducible in a large number of cell types without apparent cytopathic effects. Since its introduction 15 years ago, BacMam technology has been used in more than 70 cell types, including primary and stem cells. Table 1 lists examples of cell lines that are efficiently transduced by BacMam technology. Protein...

Cameleon gene...

+ 4 Ca CFP...

expression is transient in the absence of selection pressure and can...

mRNA translated...

Baculovirus...

Calmodulin-M13 YFP...

Endocytotic entry mRNA...

Table 1--Typical transduction efficiencies obtained using the standard PremoTM Cameleon Calcium Sensor protocol....

Cell line CHO, CHO K1, CHO M1WT3, etc. COS-7 CRL-1973 (NTERA-2) HEK 293 Transduction efficiency 50% 80% >90% 30% >90% 60% 70% >90% 80% 90% 40% 50% 40% 50% 50% 60% 40% 50% 50% 75% 40% 50% >90% 30% 40% Not readily amenable...

DNA moves to nucleus...

Cameleon gene transcribed...

Figure 1--Schematic representation of BacMam-mediated transduction and expression of the PremoTM Cameleon Calcium Sensor. The combination of fluorescent protein biosensors and BacMam del...

Document Keywords

A no - wash indicator for high - throughput calcium detection etc medialib en filelibrary pdf bioprobes bioprobes55 par 9657 file at bp55 cameleon pdf

About Ambion, Inc.

Contact

512-651-0200, 800-888-8804 (toll free)
Fax: 512-651-0190

sponsored links