Biology Reference
In-Depth Information
and adjust to pH 5.7 with potassium hydroxide (KOH), autoclave
for 20 min at 121 °C, and store at 4 °C (
see
Note 1
).
3. Miracloth (Merck KGaA, Germany).
4. Digestion buffer: 500 mM Mannitol, 5 mM 2-(
N
-morpholino)
ethanesulfonic acid (MES), adjust to pH 5.7 with KOH. Store
at 4 °C. Just prior to use, add 0.4 % (w/v) Cellulase “Onozuka”
R-10 and 0.05 % (w/v) pectolyase Y-23 (Yakult Pharmaceutical,
Japan).
5. Variable speed benchtop orbital shaker (
see
Note 2
).
6. Large-capacity preparative centrifuge with 2 × 500 mL tube
capacity capable of 800 ×
g
for sample preparation.
2.2 Protoplast
Homogenization
and Pre-FFE Fraction
1. Homogenization buffer: 1 % (w/v) dextran (Mw 200,000),
0.4 M sucrose, 10 mM disodium hydrogen phosphate
(Na
2
HPO
4
), 3 mM ethylenediaminetetraacetic acid (EDTA),
0.1 % (w/v) bovine serum albumin (BSA), 5 mM dithiothrei-
tol (DTT) (
see
Note 3
), pH to 7.1 with sodium hydroxide
(NaOH).
2. 500 mM potassium chloride (KCl), prepared in 0.4 M sucrose,
10 mM Na
2
HPO
4
, 3 mM EDTA, 1 % (w/v) dextran (Mw
200,000), 0.1 % (w/v) BSA, 5 mM DTT (
see
Note 3
), pH 7.1
with NaOH, can store at −20 °C.
3. Glass-Tefl on Potter-Elvehjem Tissue Grinders (30-50 mL
capacity), cooled on ice.
4. Preparative centrifuge with 4 × 50 mL tube capacity capable of
5,000 ×
g
for sample preparation.
5. Light microscope capable of visualizing plant cells and large
subcellular structures, minimum 40× objective.
6. Ultracentrifuge and swing-out rotor with 40 mL tube capacity
capable of 100,000 ×
g
for gradients.
7. Gradient buffer 1: 1.6 M Sucrose, 10 mM Na
2
HPO
4
, 3 mM
EDTA, pH 7.1 with NaOH, can store at −20 °C.
8. Gradient buffer 2: 1.0 M sucrose, 10 mM Na
2
HPO
4
, 3 mM
EDTA, dextran Mw 200,000 (1 % w/v), 5 mM DTT
(
see
Note 3
), pH 7.1 with NaOH, can store at −20 °C.
9. Gradient buffer 3: 0.75 M sucrose, 10 mM Na
2
HPO
4
, 3 mM
EDTA, dextran Mw 200,000 (1 % w/v), 5 mM DTT
(
see
Note 3
), pH 7.1 with NaOH, can store at −20 °C.
10. Gradient buffer 4: 0.2 M sucrose, 10 mM Na
2
HPO
4
, 3 mM
EDTA, dextran Mw 200,000 (1 % w/v), 5 mM DTT
(
see
Note 3
), pH 7.1 with NaOH, can store at −20 °C.
