Biomedical Engineering Reference
In-Depth Information
Table 2
(continued)
Name
Information/company/conditions for western blotting
Anti-p62/SQSTM1
Rabbit polyclonal antibody, Medical & Biological
Laboratories, #PM045; blocking, 10% skim milk-
TBS, incubate at room temperature for 30 min;
primary antibody reaction (dilution, 1:2,000),
incubate at room temperature for 90-120 min
Anti-ubiquitin
(1) Mouse monoclonal antibody (FK2 for multi-ubiq-
uitin), Medical & Biological Laboratories, #D058-3;
blocking, 10% skim milk-TBS, incubate at room
temperature for 30 min; primary antibody reaction
(dilution, 1:2,000), incubate at 4°C for 15 h
(2) Rabbit polyclonal antibody, DAKO, Glostrup
Denmark #Z0458; blocking, 10% skim milk-TBS,
incubate at room temperature for 30 min; primary
antibody reaction (dilution, 1:2,000), incubate at
room temperature for 90-120 min
Bold characters are essential points for recognizing target proteins by these antibodies
stored at -20°C. The 10% casein solution was diluted tenfold
with TBS (1% casein-TBS).
7. Secondary antibody solution: select appropriate secondary
antibody (see Note 7) and conditions; (a) 0.005% (v/v) goat
anti-rabbit IgG antibody (minimal cross-reactivity with other
species) conjugated to horseradish peroxidase (HRP) and
0.15% (v/v) normal goat serum in TTBS, when using a rab-
bit polyclonal antibody as the primary antibody. (b) 0.005%
(v/v) goat anti-mouse IgG antibody (minimal cross-reactivity
with other species) conjugated to HRP and 0.15% (v/v) nor-
mal goat serum in TTBS, when using a mouse monoclonal
antibody as the primary antibody. (c) 0.005% (v/v) rabbit
anti-chicken IgG antibody (minimal cross-reactivity with
other species) conjugated to HRP in TTBS, when using a
chicken polyclonal antibody as the primary antibody.
(d) 0.005% (v/v) donkey anti-goat IgG antibody (minimal
cross-reactivity with other species) conjugated to HRP and
0.15% (v/v) normal donkey serum in TTBS, when using a
goat polyclonal antibody as the primary antibody.
8. Enhanced chemiluminescent reagents: SuperSignal West
Dura Extended Duration Substrate or SuperSignal West Pico
Chemiluminescent Substrate (Pierce Biotechnology Inc./
Thermo Fisher, Rockford, IL).
1. Cell-fixative solution: 4% paraformaldehyde in PBS, pH 7.2,
prepared just before use and warmed to 37°C.
2.4. Immunofluore-
scence Analysis
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