Measurement of Autophagy in Cells and Tissues - Protein Misfolding and Cellular Stress in Disease and Aging - page 200

pH 6.8 (at 25°C), 0.05 mL of 10% SDS, 17 mL of 10% APS,
and 5 mL of TEMED) was prepared.
5. The isobutanol was removed from the polymerized running
gel by washing the top with deionized water. Residual water
was removed gently with a paper towel. The stacking gel solu-
tion was poured into the space between the glass plates, and
a comb was inserted.
6. After polymerization of the stacking gel, the comb was
removed, and the wells were washed.
7. The gel was mounted into the SDS-PAGE electrophoresis
chamber, and running buffer was added.
8. Samples (10-20 mg per lane) were loaded onto the gel. An
aliquot of 5 mL of prestained molecular weight marker was
used as a molecular weight standard.
9. The gel was electrophoresed at a constant current of 1-1.5 mA
per cm of gel width, until the pink color of the phenol red in the
markers reached the bottom of the gel (see Notes 5 and 6).

3.3. Western Blotting

1. A PVDF membrane was soaked in methanol for 1 min, trans-
ferred to Bjerrum and Schafer-Nielsen's transfer buffer, and
incubated for 5 min.
2. Four sheets of chromatography paper were soaked in the
transfer buffer.
3. After removing the glass plates and stacking gel, the running
gel was rinsed in the transfer buffer. Two sheets of chroma-
tography paper were placed on the anode plate and overlaid
with a PVDF membrane, the running gel, and two additional
sheets of chromatography paper.
4. The stack was set onto a Transblot SD Semi-Dry Transfer
Cell, and electrophoresed at 16-20 V for 1 h.
5. The gel and papers were discarded.
6. The PVDF membrane was incubated in blocking buffer at
room temperature for 30 min (see Table 2 for blocking solu-
tion suitable for each antibody).
7. The membrane was washed three times in TTBS at room
temperature for 2 min each time.
8. The membrane was incubated in primary antibody solution
(see Table 2 for appropriate dilution, incubation time, and
temperature).
9. The membrane was washed at least five times, for 5 min each
time, in TTBS at room temperature.
10. Secondary antibody reaction: the PVDF membrane was incu-
bated in secondary antibody solution for 60-90 min at room
temperature (see Note 7).