FIGURE 3.
Solubilization and purification of N-terminally tagged A2A receptors. A, purification of G2S-N-A2AR stably expressed in HEK293 cells (lane 1) was carried out in comparison with untransfected HEK293 cells (lane 2). They were monitored by Western blot (anti-A2AR) and CCD image analysis. The amount of solubilized G2S-N-A2AR is shown in lane 3, and the insoluble pellet is shown in lane 4 (both in equivalent volumes corresponding to 25 μg of original membranes). Lanes 5 and 6 represent the TEV eluate (equivalent to 10 times the original volume) and streptavidin eluate (equivalent to 100 times the original volume). B, aliquots at crucial steps of FS2-N-A2AR purification (corresponding to 32 μg of original membrane protein) (lanes 2–7, membranes, solubilized receptor, non-bound FLAG-agarose supernatant, FLAG eluate, non-bound Strep-Tactin supernatant, and final Strep-Tactin eluate, respectively) and 10 ng of FLAG-BAP control (lane 1) were quantified using scanned Western blot images (anti-FLAG). C, the yields of recovered protein complexes of typical N-tagged-A2AR purifications were calculated from pixel intensities of the respective bands, corrected for the volumes, and normalized to isolated membrane (100%, G2S-N) or solubilized aliquot (58%, FS2-N), respectively.