Biology Reference
In-Depth Information
8. Arabidopsis pollen medium (liquid, [
6
]): 18 % (w/v) sucrose,
1 mM KCl, 0.49 mM H
3
BO
3
, 1 mM MgSO
4
, 2 mM CaCl
2
,
2 mM Ca(NO
3
)
2
, pH 7. Germinate in 3 ml medium in a 25 ml
Erlenmeyer.
9. Arabidopsis pollen medium (solidifi ed, [
6
]): 18 % (w/v)
sucrose, 1 mM KCl, 0.49 mM H
3
BO
3
, 1 mM MgSO
4
, 2 mM
CaCl
2
, 2 mM Ca(NO
3
)
2
, pH 7, 1 % (w/v) agar.
10. Tobacco pollen medium [
7
]: 6 % (w/v) sucrose, 1.6 mM
H
3
BO
3
, 200
μ
M CaCl
2
, 1 mM MES adjusted with NaOH to
pH 5.5.
1. Homogenization buffer: 330 mM sucrose, 100 mM KCl,
5 mM DTT, 1 mM EDTA, 50 mM Tris adjusted with MES to
pH 7.2. Protease inhibitors are added from stock solution to
the ice-cold homogenization buffer just before use to give the
fi nal concentrations of 10
2.4 Buffers
and Solutions
for Membrane
Preparations
μ
M leupeptin, 1
μ
M pepstatin A,
M E-64 (
see
Note 6
).
2. Centrifugation buffer: 1 mM MgSO
4
, 1 mM Tris adjusted
with MES to pH 7.2.
3. Sucrose solutions: 18 % (w/w); weight in 1.8 g sucrose and fi ll
up with centrifugation buffer to 10 g. The other sucrose solu-
tions (25, 30, 34, 38, 45 % (w/w)) are prepared the same way.
1 mM PMSF, and 2
μ
1. Solution A: Dissolve subsequently the following chemicals in
750 ml distilled water in a glass beaker: 4.2 g ammonium
molybdate, 28.6 ml concentrated H
2
SO
4
, and 20 g sodium
dodecyl sulfate (SDS). Fill up to 1 l with distilled water
(
see
Note 7
).
2. Solution B: 10 % (w/v) ascorbic acid. Prepare fresh before
starting the experiment.
3. Calculate the needed total volume of the phosphate reagent
for an experiment and prepare the reagent by mixing 6 volumes
of solution A and 1 volume of solution B. Prepare fresh before
the experiment.
2.5 Solutions for ATP
Hydrolysis Assays
1. Vanadate-sensitive plasma membrane H
+
ATPase (P-ATPase):
25 mM K
2
SO
4
, 50 mM MES/Tris pH 6.8, 0.1 mM EDTA,
4 mM MgSO
4
, 100
2.6 Reaction Buffers
for ATP Hydrolysis
M Na-molybdate, 0.005 % Triton X100;
add directly to cuvettes 100 nM bafi lomycin, 1 mM sodium
azide, ±200
μ
M sodium vanadate (
see
Note 8
).
2. Bafi lomycin-sensitive vacuolar H
+
ATPase (V-ATPase): 25 mM
K
2
SO
4
, 50 mM Tris/MES pH 7.5, 0.1 mM EDTA, 4 mM
MgSO
4
, 100
μ
M Na-molybdate, 0.005 % Triton X100; add
directly to cuvettes 200
μ
M sodium vanadate, 1 mM sodium
azide, ±100 nM bafi lomycin.
μ