Biology Reference
In-Depth Information
Fig.
4
Diagram of fl oating method for growth of three plants on liquid medium in
each petri dish
MG132
100
75
50
37
25
Fig.
5
Detection of polyubiquitinated proteins in Arabidopsis Col-0 plants by
Western blot. In vivo treatment with MG132 (+) inhibited proteasome activity in
plants and thus polyubiquitinated proteins accumulated in a higher molecular
range, as detected by anti-ubiquitin antibody
5. Grind plants to a fi ne powder using liquid nitrogen to extract
proteins.
6. Add the powder to 2× SDS sample buffer that has six times the
volume of the fresh weight of the sample in a 1.5 mL tube.
7. Centrifuge at 20,000 ×
g
for 5 min at 4 °C. Transfer the super-
natant to a new tube.
8. Perform heat treatment for 3 min at 99 °C.
1. Prepare 10 % SDS-PAGE gel and apply 5
μ
L samples onto the
3.2.3 Detection of
Polyubiquitinated Proteins
gel (
see
Note 11
).
2. Separate proteins by SDS-PAGE and carry out western blot-
ting (
see
Subheading
3.1.3
).
3. Incubate the membrane with 1/5,000 anti-FK2 as fi rst anti-
body and 1/25,000 peroxidase-labeled anti-mouse antibody
as second antibody as described above (
see
Subheading
3.1.3
).
4. Detect the chemiluminescent signals for accumulation of poly-
ubiquitinated proteins as described above (
see
Subheading
3.1.3
and Fig.
5
).
