Protein
samples were denatured in the SDS sample buffer [63 mM Tris-HCl (pH
6.8) containing 0.012% bromophenol blue, 5% sucrose, and 2% SDS] for
5 min at 95 °C (nonreduced condition). To reduce protein samples,
1% beta-mercaptoethanol (2-ME) was added to SDS sample buffer and
then heat treatment was conducted. After separation of the samples
by SDS-PAGE (12.5% gel), the proteins were transferred to an Immobilon-P
Transfer Membrane (Millipore, Bedford, MA) for the western blot analysis.
The membranes were blocked in a Tris-buffered saline (pH 7.4) containing
0.1% Tween 20 (TBS-T) containing 5% skimmed milk powder (Snow Brand
Milk Products, Tokyo, Japan), incubated with anti-oxDJ-1 mAb (clone
M106, 1 μg/mL, ref (18 (link))), mouse anti-DJ-1 mAb (clone 3E8, 1 μg/mL, Medical
& Biological Laboratories, Nagoya, Japan), or anti-β-actin
mAb (AC-15) at 4 °C for 18 h, and incubated with HRP-conjugated
secondary antibodies for at least 1 h. After the incubation with Immobilon
Western (Millipore), the immunoreactivity was visualized with an LAS-4000
luminescence imager (Fujifilm, Tokyo, Japan). For silver staining,
the separated proteins were stained with the Dodeca Silver Stain Kit
(Bio-Rad Laboratories, Hercules, CA).
samples were denatured in the SDS sample buffer [63 mM Tris-HCl (pH
6.8) containing 0.012% bromophenol blue, 5% sucrose, and 2% SDS] for
5 min at 95 °C (nonreduced condition). To reduce protein samples,
1% beta-mercaptoethanol (2-ME) was added to SDS sample buffer and
then heat treatment was conducted. After separation of the samples
by SDS-PAGE (12.5% gel), the proteins were transferred to an Immobilon-P
Transfer Membrane (Millipore, Bedford, MA) for the western blot analysis.
The membranes were blocked in a Tris-buffered saline (pH 7.4) containing
0.1% Tween 20 (TBS-T) containing 5% skimmed milk powder (Snow Brand
Milk Products, Tokyo, Japan), incubated with anti-oxDJ-1 mAb (clone
M106, 1 μg/mL, ref (18 (link))), mouse anti-DJ-1 mAb (clone 3E8, 1 μg/mL, Medical
& Biological Laboratories, Nagoya, Japan), or anti-β-actin
mAb (AC-15) at 4 °C for 18 h, and incubated with HRP-conjugated
secondary antibodies for at least 1 h. After the incubation with Immobilon
Western (Millipore), the immunoreactivity was visualized with an LAS-4000
luminescence imager (Fujifilm, Tokyo, Japan). For silver staining,
the separated proteins were stained with the Dodeca Silver Stain Kit
(Bio-Rad Laboratories, Hercules, CA).
