Biology Reference
In-Depth Information
5. Detergent mixture 2: 9 mM Digitonin, 9 mM
n-dodecyl-
-
D
-
maltoside, 0.073 mM LDS, 20 % (v/v) Glycerol, 0.01 % (w/v)
Ponceau S, 15 mM Tricine, 80 mM Bis-Tris (
see
Note 1
).
β
2.3 Clear Native
LDS-PAGE from
Arabidopsis thaliana
Thylakoid Membranes
1. Thylakoid membranes from
Arabidopsis thaliana
: 1
l
frozen at liquid N
2
. Stored at −80 °C (preparation,
see
Subheading
3.1
).
2. LDS-cathode buffer: 80 mM Tricine, 15 mM Bis-Tris,
0.073 mM LDS. Assemble as 10× concentrate. Store at 4 °C
(
see
Notes 2
and
3
).
3. Anode buffer: 50 mM Bis-Tris/HCl, pH 7.0. Assemble as 10×
concentrate. Store at 4 °C (
see
Note 3
).
4. Gel buffer: 0.5 M
μ
g Chl/μl
μ
-amino caproic acid, 50 mM Bis-Tris, titrate
with HCl to pH 7.0. Assemble as 6× concentrate. Store at 4 °C
(
see
Note 3
).
5. Electrophoresis chamber: Hoefer SE400 (vertical slab gel elec-
trophoresis unit). Gel size: 180 mm × 160 mm.
6. Acrylamide solution: 30 % (w/v) acrylamide/bis acrylamide
solution (37.5:1, 2.6 % C), acts as a neurotoxin in unpolymer-
ized state. Store at 4 °C.
7. Glycerol, 50 % (v/v). Store at 4 °C.
8. TEMED:
N,N,N,N
ε
′
-tetramethyl-ethylenediamine. Store at
4 °C.
9. APS: 10 % (w/v) ammonium persulfate. Store at −20 °C.
Stable at 4 °C for up to 2 weeks.
10. Water-saturated isobutanol: 50 % (v/v) isobutanol. Store at
room temperature.
11. Peristaltic tube pump for low viscosity liquid delivery.
12. Power supply like EPS 3501 (GE Healthcare, Buckinghamshire,
UK). With ranges of voltage 35-3,500 V, current 1-400 mA,
power 1-200 W, time 0:01-500 h, volt hours 1-500,000 Vh,
milliampere-hours 1-25,000 mAh.
2.4 Analysis of
Thylakoid Membrane
Complexes
1. Molecular weight standard: Invitrogen NativeMark, unstained
protein standard, IgM Hexamer, IgM Pentamer, Apoferritin
band 1, Apoferritin band 2,
-phycoerythrin, Lactate
Dehydrogenase, Bovine Serum Albumin, Soybean Trypsin
Inhibitor, with molar masses of 1.236 kDa, 1.048 kDa, 720 kDa,
480 kDa, 242 kDa, 146 kDa, 66 kDa, 20 kDa, respectively.
β
2. White light scanning: Apple Color OneScanner 1200/30.
3. Fluorescence scanning: Li-COR Odyssey, Imaging system
(Li-COR Biosciences). Excitation wavelength 680 nm,
Emission wavelength 694 nm.
4. Colloidal Coomassie stain, 0.25 % (w/v) Coomassie G250,
50 % (v/v) Methanol, 10 % (v/v) Acetic acid.
