Proteins
were analyzed by SDS–PAGE in 12% gels under fully denaturing
conditions (boiling of the samples for 10 min prior to loading) or
semi-denaturing conditions (no boiling of the samples prior to loading).
In-gel fluorescence was analyzed on a UVP ChemiDoc-It2 Imaging System
equipped with a cooled CCD camera and a GFP filter, after exposure
for about 3 s, following SDS–PAGE analysis under semi-denaturing
conditions. For Western blot analysis, proteins were transferred to
polyvinylidene fluoride membranes (Merck) for 45 min at 12 V on a
semi-dry blotter (Thermo Fisher Scientific). Membranes were blocked
with 5% (w/v) non-fat dried milk in Tris-buffered saline containing
0.1% (v/v) Tween-20 (TBST) overnight at 4 °C. After three washes
with TBST, they were incubated with the appropriate antibody dilution
in TBST containing 0.5% (w/v) non-fat dried milk at room temperature
for 1 h. Specifically, the utilized antibodies were a mouse monoclonal
anti-polyhistidine antibody (Sigma) at 1:3000 dilution (conjugated
with horseradish peroxidase), a mouse monoclonal anti-GFP antibody
(Takara) at 1:20,000 dilution, or a mouse monoclonal anti-FLAG antibody
(Sigma) at 1:2500 dilution, with a horseradish peroxidase-conjugated
goat anti-mouse IgG secondary antibody at 1:4000 dilution. The proteins
were visualized using a ChemiDoc-It2 Imaging System (UVP), after triple
membrane washing with TBST.
were analyzed by SDS–PAGE in 12% gels under fully denaturing
conditions (boiling of the samples for 10 min prior to loading) or
semi-denaturing conditions (no boiling of the samples prior to loading).
In-gel fluorescence was analyzed on a UVP ChemiDoc-It2 Imaging System
equipped with a cooled CCD camera and a GFP filter, after exposure
for about 3 s, following SDS–PAGE analysis under semi-denaturing
conditions. For Western blot analysis, proteins were transferred to
polyvinylidene fluoride membranes (Merck) for 45 min at 12 V on a
semi-dry blotter (Thermo Fisher Scientific). Membranes were blocked
with 5% (w/v) non-fat dried milk in Tris-buffered saline containing
0.1% (v/v) Tween-20 (TBST) overnight at 4 °C. After three washes
with TBST, they were incubated with the appropriate antibody dilution
in TBST containing 0.5% (w/v) non-fat dried milk at room temperature
for 1 h. Specifically, the utilized antibodies were a mouse monoclonal
anti-polyhistidine antibody (Sigma) at 1:3000 dilution (conjugated
with horseradish peroxidase), a mouse monoclonal anti-GFP antibody
(Takara) at 1:20,000 dilution, or a mouse monoclonal anti-FLAG antibody
(Sigma) at 1:2500 dilution, with a horseradish peroxidase-conjugated
goat anti-mouse IgG secondary antibody at 1:4000 dilution. The proteins
were visualized using a ChemiDoc-It2 Imaging System (UVP), after triple
membrane washing with TBST.