Biology Reference
In-Depth Information
2.2 In Vivo MG132
Treatment
and Detection of
Ubiquitinated Protein
Arabidopsis thaliana
(Col-0).
2.2.1 Plant Material
1. MS medium: 4.3 g/L Murashige and Skoog (MS) basal salt
mixture, vitamin mixture (nicotinic acid 0.5 mg/L, pyridoxine
HCl 0.5 mg/L, thiamine HCl 0.1 mg/L, glycine 2.0 mg/L,
myo-inositol 100 mg/L), 2 % sucrose, and 0.25 % gellan gum,
adjust pH 5.7 with KOH.
2. 1/2 MS liquid medium: 2.15 g/L Murashige and Skoog basal
salt mixture, vitamin mixture, 1 % sucrose, adjust pH 5.7 with
KOH.
3. MG132 stock solution: 10 mM MG132 in DMSO.
2.2.2 Growth Medium
1. 2× SDS sample buffer: 4 % SDS, 10 % 2-mercaptoethanol,
125 mM Tris-HCl, pH 6.8, 20 % glycerol and 0.002 % bro-
mophenol blue (BPB).
2. 10 % SDS-polyacrylamide gel.
3. SDS electrophoresis running buffer: 50 mM Tris-HCl, pH 8.9,
384 mM glycine and 0.1 % SDS.
4. Transfer buffer: 39 mM glycine, 48 mM Tris, 0.0375 % SDS
and 20 % methanol.
5. PBS-T buffer: 80 mM Na
2
HPO
4
, 20 mM NaH
2
PO
4
, 100 mM
NaCl and 0.1 % Tween-20.
6. Blocking buffer: PBS-T with 5 % skimmed milk.
7. Antibody: anti-multiubiquitin chains antibody (FK2) (Nippon
BioTest laboratories Inc., Tokyo, Japan), peroxidase-labeled
anti-mouse IgG antibody (GE Healthcare, Little Chalfont,
UK).
8. Detection solution: Immobilon Western Chemiluminescent
HRP Substrate (Millipore, Massachusetts, USA).
2.2.3 SDS-PAGE
and Western Blotting
3
Methods
1. Grow Agrobacterium transformed with each binary vector in
LB medium supplemented with 50
3.1 Proteasome-
Dependent
Degradation Assay
with Tobacco Transient
Expression System
g/mL Kanamycin up to
1.0 OD
600
and harvest 1 mL Agrobacterium medium into
1.5 mL tube (
see
Note 5
).
2. Centrifuge the Agrobacterium at 2,000 ×
g
for 3 min in room
temperature and remove the supernatant.
3. Resuspend and wash with 1 mL infi ltration buffer and centri-
fuge again under the same conditions.
μ
3.1.1 Agrobacterium
Culture and Infi ltration
