Figure 3.

(A) A schematic of nonspecific fluorogenic photoaffinity labeling (PAL) of whole cells. The nitrene intermediate resulting from the photoconversion of an azide to an amine is reactive enough to insert into bonds of nearby biomolecules. The reaction simultaneously turns-on fluorescence and covalently links the probe to the biomolecule. (B) Gel electrophoresis of CHO-cell lysate demonstrating fluorogenic PAL using NBD-azide. (Similar results were observed with DCDHF-P-azide, data not shown.) The left panel shows the stained protein, imaged with white light; the right panel is fluorescence from NBD-amine photochemically cross-linked to proteins, imaged using 488 nm. The left lane in the gel (+PAL) is protein covalently labeled with NBD-amine by PAL. The right lane (−PAL) is a control performed by mixing into the cell solution preactivated NBD-amine, which is fluorescent but cannot participate in the covalent PAL bioconjugation reaction. The fluorescence signal in the control lane was significantly lower. The blurry fluorescence on the bottom of the gel is from the unbound dye at the front edge; equal brightness in both lanes indicates equal dye concentration in the PAL and control.