Biology Reference
In-Depth Information
5. Phase buffer: 250 mM sucrose, 5 mM KCl, and 5 mM PO
4
−
buffer. Weigh 8.56 g sucrose, transfer it to a 100 mL volumet-
ric fl ask, add 2.5 mL 200 mM KCl and 200 mM PO
4
−
buffer,
fi ll up ad 100 mL with H
2
O.
2.4
Tonoplast
1. 1 M Tris solution.
2. 200 mM Tris solution.
3. Tonoplast resuspension medium: 250 mM mannit, 100 mM
tricine, 3 mM MgSO
4
, 3 mM EGTA, pH 7.5. Adjust the pH
with 1 M Tris-solution.
4. Gradient solution: 28 % sucrose, 5 mM HEPES, and 2 mM
DTT, pH 7.5. Adjust the pH with 200 mM Tris solution.
5. Dilution medium: 3 mM MgSO
4
and 50 mM HEPES-NaOH,
pH 7.0.
6. Storage medium: 40 % Glycerol, 1 mM EDTA, 10 mM
HEPES, pH 7.0. Adjust the pH with 0.1 M NaOH.
2.5 Protein
Quantifi cation
1. Bradford [
42
] reagent: Dissolve 50 mg Coomassie-Brilliant
Blue G in 50 mL ethanol, mix it with 100 mL of 85 % phos-
phoric acid. Pour the acid solution carefully in H
2
O and fi ll it
ad 1 L. Filter the solution into a brown bottle and store it at
room temperature.
2. Dilutor: 0.9 % NaCl and 0.01 % Triton X-100 in Milli Q water
(freshly prepared).
3. Bovine serum albumin (BSA) Standard solution: 0.02 % BSA
(5
L = 1 mg protein).
4. UV/VIS-Spectrophotometer.
μ
2.6 Membrane-
Washing
1. Wash buffer: 50 mM HEPES-KOH buffer, pH 7.0, 1 mM
EDTA, 0.01 % Triton X-100 and 150 mM KCl.
All glass plates used for casting gels should be degreased before
use. Mini-Protean unit (Bio-Rad, Germany) and SE 250 vertical
unit (GE Healthcare, Germany) are suitable for the electrophoresis
types described but the protocols can be also adapted to bigger
systems. Power supplies should allow for a voltage around 100-
500 V and a current around 200-500 mA. A peristaltic pump and
a gradient mixer are required to cast acrylamide gradient gels.
2.7
Electrophoresis
1. Ready-made acrylamide/Bis solution (AB-solution) (Rotiphorese
®
Gel 30 (37, 5:1)) (Carl Roth, Germany). Store at 4 °C.
2. Ammonium persulfate (APS): 10 % solution in water (
see
Note 2
).
3. N,N,N,N
-tetramethyl-ethylenediamine (TEMED) (Sigma-
Aldrich, Germany). Store at 4 °C.
4. Pre-stained protein ladder (Thermo Scientifi c, Germany).
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