Intracellular imaging
of HaloTag-modified amylase using immunofluorescence
microscopy. (A) Two HaloTag–amylase proteins (HaloTag A and
HaloTag B) were constructed, as the exact translational start site
for salivary amylase has not been identified. (B) Expression of nonconjugated
HaloTag (Halo) and both HaloTag A and B proteins was examined. The
top band represents the HaloTag complex, whereas the smaller band
is indicative of pure HaloTag without amylase attached. (C) Halo,
HaloTag A, and HaloTag B were labeled with a HaloTag ligand (TMR-Green),
and secretory granules were labeled with an anti-amylase antibody
(shown in red). Both HaloTag A and B show colocalization with endogenous
amylase, indicating that both were in secretory granules. Scale bar
= 10 μm. Reprinted with permission from ref (92). Copyright 2013 the American
Physiological Society.