FLAG-tagged receptors

By expressing FLAG-tagged receptors, the amount of receptor on the cell surface in the absence or presence of ligands of interest can be measured by ELISA with anti-FLAG Ml antibodies. A more advanced technique using confocal microscopy and staining of cells prior and post-permeabilization of the cell membrane also allows to visually track the receptor and assess the internalized receptor fraction. 

Untransfected Chemokine no tag Receptor C FLAG Receptor N FLAG Chemokine C HA... 

Expression constructs were designed with the receptor sequence cloned into a pFastBac vector. A schematic of the receptor construct in the vector is shown in Fig. 2A. This construct utilizes a GP64 or poM promoter to drive receptor expression, an HA signal sequence to enhance localization in the Sf9 ceU membrane and a C-terminal Prescission protease (PP) cleavage site followed by FLAG and His tags which permit detection and purification, respectively. Restriction sites before and after the receptor sequence facilitate transfer of sequences between plasmids with different promoters, signal... 

In addition to phosphorylation, the activity of receptors is controlled by the formation of homo- or heteromers. TR-FRET is a perfect tool to follow receptor activation on the membranes exemplified by opioid receptor [57], dopamine receptor [58, 59], histamine receptor [60], adrenoceptors [60, 61], EL-13 [62, 63], chemokine receptors [64, 65], and receptors related to EBV infection [66]. Most commonly, the assays have been accomplished with receptors coexpressed with suitable tags, such as c-myc, polyhistidine, glutatione S transferase (GST) or FLAG, and using europium- and APC-labeled antibodies. 

---