Preparation Of The Plasma Membrane Fraction

The method is described here as it is applied to the isolation of plasma membranes from human multiple myeloma RPMI 8226 cells grown in suspension and human breast cancer MCF-7 cells cultured in monocellular layers. We thank Dr. R. Fenton of the Greenebaum Cancer Center, University of Maryland School of Medicine, for the multiple myeloma cells, and Dr. K.H. Cowan of the Eppley Institute, University of Nebraska Medical Center, for the MCF-7 cell line.

6.2.1 Materials

Cationic colloidal silica (Ludox CL) and polyacrylic acid (100,000 average molecular weight fraction) were obtained from Sigma Aldrich (St. Louis, Mo.). Nycodenz, Laemmli buffer, growth media, phosphate-buffered saline (PBS), and other chemicals were obtained from either Bio-Rad (Hercules, Calif.) or Sigma Aldrich.

Membrane coating buffer A: 20 mM MES, 150 mM NaCl, 800 mM sorbitol, pH 5.3

Membrane coating buffer B: 20 mM MES, 135 mM NaCl, 0.5 mM CaCl2,

1 mM MgCl2, pH 5.3 Polyacrylic acid solution: 10 mg/ml polyacrylic acid in membrane coating buffer A or B, pH 6.5. Lysis buffer: 2.5 mM imidazole pH 7

6.2.2 Cell Suspensions

The RPMI 8226 cells are grown in RPMI 1640 media and heat-inactivated fetal calf serum8 in 150 cm2 cell culture flasks at 37°C under 5% CO2 The RPMI cells are recovered by centrifugation at 900g. About 1.5 g wet weight cells are washed in 50 ml plasma membrane coating buffer A (see above) and added dropwise into 15 ml of a suspension of 10% (by volume) cationic colloidal silica in the same membrane coating buffer. The suspension is rocked gently on ice for 15 minutes. The silica-coated cells are then sedimented at 900g for 5 minutes, and the supernatant suspension is removed by decantation.

The silica-coated cells are resuspended in 50 ml buffer A and sedimented once more at 900g for 5 minutes to remove any excess silica. The cells are resuspended in 7 ml buffer solution A, added dropwise to 15 ml of polyacrylic acid solution (see above), and placed on ice for 15 minutes with gentle rocking. The silica and polyacrylic acid coated cells are then sedimented at 900g for 5 minutes, and the supernatant is removed by decantation. The cells are washed with 50 ml plasma membrane coating buffer A, recovered by centrifugation at 900g for 5 min, and placed in 50 ml lysis buffer with 0.5 ml of Sigma protease inhibitor cocktail and left on ice for 30 minutes.

The swollen cells are then lysed using nitrogen cavitation at 1500 psi. The cell lysate is centrifuged at 900g for 30 minutes to sediment the silica-coated plasma membrane pieces, along with nuclei. The pellet is resuspended in 10 ml lysis buffer and diluted with an equal amount of 100% Nycodenz in lysis buffer. This solution is layered over a 70% Nycodenz lysis buffer solution in six 4 ml centrifuge tubes. Lysis buffer is then layered onto each tube to fill it to the top, and the tubes are spun at 60,000g in an SW60Ti rotor for 23 minutes. The silica-coated plasma membranes are collected in the pellet, leaving the nuclei at the 50%:70% Nycodenz interface. The supernatant is drawn off, and the silica-coated plasma membrane pellets are resuspended in lysis buffer and centrifuged at top speed on a bench top microfuge to remove the excess Nycodenz. The pellet is washed in this way two more times with lysis buffer and then three times with 100 mM Na2CO3 (pH 11.4) for a total of six washes.

The purified plasma membrane proteins are then recovered from their silica coating by suspension directly in Laemmli loading buffer, incubation in a 60°C water bath for 30 minutes, sonication 5 times for 10 seconds each at maximum setting, and incubation in the 60°C water bath for an additional 30 minutes. Finally the suspension is spun at maximum speed in a microfuge for 15 minutes to pellet the silica. The supernatant, which now contains the solubilized plasma membrane proteins, is drawn off and stored at 80°C, and the pellet is discarded.

6.2.3 Cells Growing in Monolayers

MCF-7 cells were grown to confluence in 150 cm2 flasks with Improved Minimal Essential Medium containing 5% fetal calf serum and antibiotics at 37°C and 5% CO2.9 Typically twelve flasks are processed in parallel.

The medium is drawn off and the adherent cells are washed twice with PBS containing 1 mM MgCl2 and 1 mM CaCl2 and then with plasma membrane coating buffer B (see above). The cells are then coated with 15 ml of a 5% suspension of cationic colloidal silica in the membrane coating buffer B and left on ice for 1 minute. The silica suspension is removed, followed by a wash with membrane buffer solution B to remove excess silica.

The coated cells are treated with 15 ml of a 10 mg/ml solution of polyacrylic acid in membrane buffer B, pH 6.0 to 6.5, and left on ice for 1 minute. The polyacrylic acid solution is removed, and the cells are washed with 15 ml membrane buffer B.

The adherent cells are washed quickly with 15 ml lysis buffer (see above), and then 15 ml lysis buffer containing 0.5 ml Sigma protease inhibitor cocktail is added to the flasks, which are left on ice for 30 minutes. Once the cells have swollen, the flasks are placed on a bench top and allowed to reach room temperature.

The apical part of the plasma membranes, which has been coated by silica and polyacrylic acid, is sheared from the rest of the cell by pipetting the lysis buffer in each flask up and down over the cells. The sheared coated apical membrane sheets are drawn off with a pipette and treated as described in the previous section, finally solubilizing the proteins in Laemmli loading buffer.

The basolateral portions of the plasma membranes are still attached to the cell culture surface. Following lysis and removal of the apical fragments, any remaining lysis buffer is poured off and 15 ml of 5 M NaCl solution is added to each flask.

After the flask is rocked for 5 minutes at room temperature, the NaCl solution is poured off and 15 ml PBS containing 10 mM EDTA is added to each flask and rocked for 5 minutes at room temperature. The PBS-EDTA solution is poured off, and the flask is washed quickly with 15 ml of 100 mM Na2CO3, pH 11.4, to remove excess PBS-EDTA. Then 15 ml of 100 mM Na2CO3, pH 11.4, is again added to each flask and incubated for 5 minutes at room temperature with rocking. This is poured off and a third 15 ml aliquot of 100 mM Na2CO3 is added to each flask.

The basolateral membranes are scraped from the bottom of the flask with a cell scraper and spun at 14,000g in an SW28 rotor for 20 minutes. The purified baso-lateral fragments are recovered in the pellet, resuspended in a minimal amount of 100 mM Na2CO3, and transferred to a 1.5 ml microfuge tube and spun at maximum speed in a microfuge for 20 minutes to pellet the plasma membranes. The basolateral plasma membrane pellet can then be solubilized in 2% SDS or directly in Laemmli loading buffer, augmented by sonication and a 60°C water bath as described above.

Supplements For Diabetics

Supplements For Diabetics

All you need is a proper diet of fresh fruits and vegetables and get plenty of exercise and you'll be fine. Ever heard those words from your doctor? If that's all heshe recommends then you're missing out an important ingredient for health that he's not telling you. Fact is that you can adhere to the strictest diet, watch everything you eat and get the exercise of amarathon runner and still come down with diabetic complications. Diet, exercise and standard drug treatments simply aren't enough to help keep your diabetes under control.

Get My Free Ebook

Post a comment