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Last updated date: Jul 9, 2024 Views: 29 Forks: 0
Tonoplast membrane preparation
Adapted from (Barkla et al., 1999)
Prepare buffers:
Homogenization buffer:
| Final Concentration | 250 ml (1 extraction) | 500 ml (2 extractions) |
Mannitol | 400 mM | 18.2 g | 36.4 g |
Glycerol | 10% (w/v) | 25 g | 50 g |
Tris | 30 mM | 3.5 g | 7 g |
BSA | 0.5% (w/v) | 1.25 g | 2.5 g |
EGTA | 5 mM | 0.35 g | 0.7 g |
BHT stock | 0.5 mM | 0.5 ml | 1 ml |
Dibucaine stock | 0.25 mM | 0.1 ml | 0.2 ml |
MgSO4 stock | 5 mM | 2.5 ml | 5 ml
|
Just before use add:
| |||
PMSF | 1 mM | 2.5 ml | 5 ml |
DTT(Note 3) | 2 mM | 0.077 g | 0.154 g |
Benzamidine | 1 mM | 0.039 g | 0.078 g |
K-metabisulfite | 26 mM | 1.4 g | 2.8 g
|
Set pH to 8.0 with H2SO4 |
| ||
PVP-10 (Note 4)
| 5% (w/V) | 12.5 g | 25 g |
Resuspension buffer:
|
| 100 ml (1 extraction) | 200 ml (2 extractions) |
Mannitol | 400 mM | 7.28 g | 14.56 g |
Glycerol | 10 % | 10 g | 20 g |
Tris/MES pH 8 (0.3 M stock) | 6 mM | 2 ml | 4 ml
|
Just before use add:
| |||
DTT(Note 3) | 1.3 mM | 0.02 g | 0.04 g
|
Set pH to 8.0 with Tris or MES crystals |
22 % Sucrose in resuspension buffer:
2 tubes: 25 ml resuspension buffer plus 5.995 g sucrose
4 tubes: 44 ml resuspension buffer plus 10.5 g sucrose
6 tubes: 66 ml resuspension buffer plus 15.83 g sucrose
EGTA prevents membrane aggregation and binds heavy metals
K-metabisulfite prevents oxidative reactions
PVP-10 binds phenolic compounds released from the vacuole
BSA serves as a substrate for protease activity and binds free fatty acids
BHT (butylated hydroxytoluene) inhibits lipid breakdown
PMSF (phenylmethylsulphonyl fluoride) inhibits proteolysis
Dibucaine inhibits Phosphatase A
Stock solutions:
Tris/MES pH 8, 0.3 M:
Tris (MW: 121.1 g/mol): 3.633 g/100 ml
add MES until pH 8
MgSO4, 0.5 M:
MgSO4 (MW: 246.48 g/mol): 12.324 g/100 ml
PMSF (phenylmethylsulphonyl fluoride), 0.1 M:
PMSF: 0.871 g/50 ml isopropanol (store at -20°C)
BHT (butylated hydroxytoluene), 250 mM:
BHT: 0.551 g/10 ml isobutanol (store at 4°C)
Dibucaine:
0.25 g/1 ml isobutanol (store at 4°C)
Notes:
Purification of microsomes:
Purification of tonoplast vesicles:
References:
Barkla, B. J., Vera-Estrella, R., Maldonado-Gama, M., & Pantoja, O. (1999). Abscisic acid induction of vacuolar H+-ATPase activity in Mesembryanthemum crystallinum is developmentally regulated. Plant Physiology, 120(3), 811–819. https://doi.org/10.1104/pp.120.3.811
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