Biology Reference
In-Depth Information
2
Materials
2.1 Proteasome-
Dependent
Degradation Assay
with Tobacco Transient
Expression System
1.
Nicotiana benthamiana
.
2.
Rhizobium radiobacter
(GV3101 pMP90).
2.1.1 Tobacco
and Agrobacterium
p35S:ATL31-FLAG (pGWB11), p35S:Myc-14-3-3× (pEarley-
Gate203), p35S:GFP (pMDC43), p35S-p19 (pBIC) (
see
Note 1
).
2.1.2
Binary Vector
1. LB: 0.5 % yeast extract, 1 % Polypeptone, and 1 % NaCl.
2. Antibiotics: Kanamycin.
3. Infi ltration buffer: 10 mM MgCl
2
and 10 mM MES-NaOH
(pH 5.6).
4. 150 mM Acetosyringone (3,5
2.1.3 Culture Medium
and Infi ltration Buffer
-hydroxy-
acetophenone) dissolved in dimethyl sulfoxide (DMSO) (
see
Note 2
).
′
-dimethoxy-4
′
1. Protein extraction buffer: 50 mM Tris-HCl, pH 7.5, 2 mM
ATP, 5 mM MgCl
2
, 10 mM 2-mercaptoethanol, and 20 %
glycerol (
see
Note 3
).
2. 10 mM MG132 dissolved in DMSO (
see
Note 4
).
2.1.4 Protein Extraction
and MG132 Treatment
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-Myc antibody from mouse (PL14, MBL,
Nagoya, Japan), anti-GFP antibody from mouse (JL-8,
Clontech, California, USA), peroxidase-labeled anti-mouse
IgG antibody (GE Healthcare, Little Chalfont, UK).
8. Detection solution: Immobilon Western Chemiluminescent
HRP Substrate (Millipore, Massachusetts, USA).
2.1.5 SDS-PAGE
and Western Blotting