Measurement of Autophagy in Cells and Tissues - Protein Misfolding and Cellular Stress in Disease and Aging - page 193

ethylenediamide (TEMED) (see Note 5 for ready-made gel
and recognition of LC3-I and LC3-II).
2. Ammonium persulfate (APS): 10% (w/v) in water, prepared
immediately before use.
3. Water-saturated isobutanol: Equal volumes of water and isob-
utanol were shaken in a glass bottle and allowed to separate.
The top layer was stored at room temperature.
4. Running buffer: 25 mM Tris, 192 mM glycine, and 0.1%
(w/v) SDS. Do not adjust pH. Store at room temperature.
5. Prestained molecular weight markers, broad range (6-175 kDa)
(New England Biolabs, Beverly, MA): The solution contained
0.01% (w/v) phenol red and 0.01% (w/v) bromophenol blue
(see Note 6 for recognition of LC3-I and LC3-II).
6. Apparatus for SDS-gel electrophoresis: for example, NA-1030
(Nihon-Eido, Tokyo, Japan) for mini-wide gels (1.0 mm
thick, 200 mm wide, 105 mm high, suitable for 16-20 sam-
ples per gel).

2.3. Western Blotting

1. Primary antibody solution: 0.1-1 mg/mL primary antibody
(see Table 2 ), 1% (w/v) fraction V bovine serum albumin
(BSA) (or 10% skim milk), 20 mM Tris-HCl, pH 7.5 at 25°C,
150 mM NaCl (TBS), and 0.1% NaN 3 .
2. Bjerrum and Schafer-Nielsen's transfer buffer: 48 mM Tris,
39 mM glycine, 20% methanol (analytical grade; Wako), pH
9.0-9.4, depending on reagent purity. Do not adjust pH; if
lower than pH 9.0, prepare again.
3. Polyvinylidene difluoride (PVDF) membrane (Durapore
membrane GV, pore size 0.22 mm; Millipore, Bedford, MA)
and chromatography paper (cat no 590; Advantec Japan,
Tokyo, Japan).
4. Transblot SD Semi-Dry Transfer Cell (BioRad, Hercules,
CA) for transferring proteins from polyacrylamide gels onto
PVDF membranes. Transfer proteins from a single gel to a
single PVDF membrane per cell. Avoid transferring proteins
from two or more gels onto PVDF membranes per cell.
5. TTBS: 20 mM Tris-HCl, pH 7.5 at 25°C, 150 mM NaCl,
and 0.05% (v/v) polyoxyethylene sorbitan monolaurate
(Tween 20).
6. Blocking buffers: (a) 10% (w/v) nonfat dry milk in TTBS (or
TBS: 20 mM Tris-HCl, pH 7.5 at 25°C, 150 mM NaCl),
(b) 5% BSA (Fraction V; Sigma-Aldrich)-TBS, (c) 1% BSA-
TBS, or (d) 1% casein-TBS. For preparing 10% casein solu-
tion, 100 mM maleic acid and 150 mM NaCl were dissolved
and adjusted to pH 7.5 using solid NaOH. Thereafter, casein
was added and dissolved with mild heating. This solution was