Journal: Nature protocols
Article Title: Fluorescent labeling of tetracysteine-tagged proteins in intact cells
Figure Lengend Snippet: Specific labeling with FlAsH can only be seen after appropriate washing. Confocal images of a human A 2A -adenosine receptor construct with the tetracysteine motif CCPGCC in intracellular loop 3 and CFP at the C-terminus at position Gly-340 (A 2A ) transiently expressed in HEK293 cells. Cells were imaged 48 h after transfection. The top row shows cells after labeling with 500 nM FlAsH according to the protocol without washing (thus, without Steps 11–13). The bottom row shows the same cells 10 min after addition of 250 µM EDT according to the protocol. The left column shows images excited at 430 nm and emission from 470–550 nm (i.e., CFP fluorescence); the middle column shows the same cells excited at 514 nm and emission measured from 530–600 nm (i.e., FlAsH fluorescence). After addition of EDT, the fluorescence intensity of FlAsH-labeled cells was 15- to 20-fold over background of nontransfected neighboring cells. Note that the mere addition of EDT does not represent the full washing procedure and that better background reduction is obtained with the full procedure. The right column represents the transmission images of cells. The white scale bar = 10 µm.
Article Snippet: Xma I (NEB, 5,000 U, cat. no. R0180S) Sma I (NEB, 2,000 U, cat. no. R0141L) TC-FlAsH (Invitrogen, labeling kit, cat. no. T34561) Invitrogen is the only commercial supplier, but we have successfully used self-synthesized reagents (our FlAsH-EDT2 stock is 1 mM in DMSO; stock solutions are stored at − 20 °C) TC-ReAsH (Invitrogen, labeling kit, cat. no. T34562) Invitrogen is the only commercial supplier, but we have successfully used self-synthesized reagents.
Techniques: Labeling, Construct, Transfection, Fluorescence, Transmission Assay