Tonoplast membrane preparation

Tonoplast membrane preparation 

Adapted from (Barkla et al., 1999)

Prepare buffers:

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:

1. Precool all buffers and materials to 4°C and work in the cold room or on ice at all times!!!
2. If you have more than two extractions and need to extract over several days, prepare the buffers fresh on each day!
3. If you are interested in post-translational modifications, reconsider the use of DTT and look into alternatives.
4. PVP-10 is not added to the buffer but directly onto the leaf material before blending.

Purification of microsomes:

• Harvest 75 g rosette leaf material from plants grown under short day conditions. 
• Put plant material into blender with 250 ml homogenization buffer. Add the PVP-10.
  • do 3 pulses of appr. 10 seconds at full speed with 10 seconds in between after you mixed buffer and plant material with some short pulses.
• Filter the solution through two layers of Miracloth.
• Distribute the solution equally among centrifuge tubes (appr. 8 tubes)
• Centrifuge at 10.000 g for 20 minutes (acceleration = max; deceleration = max).
• Pour the supernatant into an ultracentrifuge tubes and centrifuge at 100.000 g for 45 minutes (acceleration = max; deceleration = max).
  • be sure not to transfer anything of the pellet (green stuff)
  • the pellet can be discarded.
• Resuspend the pellets in a minimum volume of resuspension buffer.
  • resuspend pellet of 1 tube with 500 µl, then transfer this 500 µl to the next tube and add 500 µl to the first tube to obtain any remaining microsomes. Continue until all pellets are resuspended. In total, use a maximum volume of 3 ml.
  • try to avoid foam!
• Transfer the whole volume to a 3 ml glass tissue homogenizer.
  • homogenize to get rid of any remaining, not resuspended pieces and to reduce foam.
  • you can take an aliquot (15 µl) for determination of fold enrichment of tonoplast vesicles via Western Blotting.

Purification of tonoplast vesicles:

• Transfer the microsomes on top of a 9.5 ml 22% sucrose in resuspension buffer in ultracentrifugation tubes.
  • use a 3 ml plastic Pasteur pipette to layer the microsomes on the sucrose gradient
  • use tubes for a swinging-bucket rotor.
  • distribute the microsomes equally among two or four tubes.
  • do not transfer foam!
• Centrifuge at 97.000 g for 2 hours (acceleration = min; deceleration = min).
  • use a swinging-bucket rotor.
• Transfer the 0/22 % Sucrose interphase of all tubes to one ultracentrifuge tube filled half way with resuspension buffer.
  • Use a fine tip 3 ml plastic Pasteur pipette to get to the interphase.
  • The interphase should appear as a fluffy layer.
  • Do not take up too much sucrose.
• Centrifuge at 150.000 g for 45 minutes (acceleration = max; deceleration = max).
• Remove and discard supernatant carefully.
  • In order not to lose the pellet do not pour out the supernatant!
• Very carefully resuspend the pellet in 150-300 µl of resuspension buffer.
• Transfer pellet into a 1 ml glass tissue homogenizer. Carefully homogenize for a few seconds on ice.
• Transfer the 50 ul aliquots of the homogenate into 1.5 ml tubes to avoid multiple freeze/thaw cycles.
  • you can take an aliquot (15 µl) for determination of protein concentration via Bradford and fold enrichment of tonoplast vesicles via Western Blotting.
• Freeze samples in liquid nitrogen and store at -80°C.

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