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Protocols > Plasma membrane fraction

Protocol for the isolation of plasma membranes from plant tissues. Protocol courtesy of Dr. Masayoshi Maeshima, Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences Nagoya University, Nagoya, Japan. In the original protocol, 2-4 mg from 200 g of mung bean hypocotyls of 3.5-day-old seedlings were homogenized and used. The plasma membrane accounts for 10% of the total membrane fraction (crude membrane fraction). The ER membrane is rich in the crude membrane fraction. This protocol can be scaled-down. If 50-100 g of plant tissue is used, the first precipitate should be suspended with 2 ml of sucrose/KP/DTT, followed by mixing the suspension with 2 ml of the phase separation solution.

Protocol

Filter tissue homogenate and subsequently centrifuge at 10,000 xg for 15 min.
Collect the supernatant and centrifuge it at 150,000 xg for 30 min.
Suspend the obtained precipitate with 10 ml of sucrose/KP/DTT.
Mix the suspension strongly for 30 min at 0°C with 10 ml of the phase separation solution.
** The ratio of the crude membrane suspension to Phase separation solution is 1:1 (10 ml to 10 ml).
Centrifuge tubes at 4,500 xg for 4 min.
Mix 10 ml of the phase separation solution with 10 ml of sucrose/KP/DTT to obtain the lower phase (pure lower phase).
** The ratio of sucrose/KP/DTT to Phase separation solution is 1:1 (10 ml to 10 ml).
After centrifugation, the plasma membrane is recovered in the upper phase (PEG phase).
Mix the obtained upper phase with the pure lower phase.
Mix the suspension strongly for 30 min at 0°C and then centrifuged at 4,500 xg for 4 min.
The plasma membrane is recovered in the upper phase.
Add three volumes of sucrose/KP/DTT to the upper phase. Mix the suspension well and subsequently centrifuge at 100,000 xg for 30 min.
Suspend the precipitate with 1 ml of sucrose/KP/DTT. You can use the suspension as plasma membrane fraction.

Solutions Required

PHASE SEPARATION SOLUTION

PEG 3540 (SIGMA P-3640)		3.0 g
DextranT500 (Pharmacia)		3.0 g
Sucrose/KP/DTT		28.0 ml
300 mM NaCl/sucrose/KP/DTT		4.8 ml

IMPORTANT: Store at 0°C (on ice) in four 50 ml Falcon tubes.

STOCK SOLUTIONS

1. 100 mM K-phosphate buffer, pH 7.8 (200 ml)

2. 0.25 M sucrose, 10 mM K-phosphate buffer, pH 7.8, 1 mM DTT (sucrose/KP/DTT) (500 ml)

3. 300 mM NaCl in sucrose/KP/DTT (100 ml)

Scheme