Extraction and solubilization experiments of plant plasma membrane proteins
In all living cells the plasma membrane (PM ) exists as an interface between the cytoplasm and the environment and is one of the most complex and differentiated membranes. Since many of these proteins are involved in essential cellular functions such as cell signaling, osmoregulation, nutrition, and metabolism, the identification and functional analysis of membrane proteins (either extrinsic or intrinsic) is a crucial challenge. The source of this experiment is the "Guide to Plant Proteomics Experiments" [French] H. Tillmant, M. Zivi, C. Damerweil, V. Mitchen, eds.
Operation method
Extraction and solubilization of plant plasma membrane proteins
Materials and Instruments
Arabidopsis thaliana Move 3.1 Separation of Plasma Membranes For more product details, please visit Aladdin Scientific website.
Ultrapure water EGTA storage solution NaF storage solution Wash buffer (WBSC ) Microparticle buffer
Waring Stirrer Cell Breaker
1. Separation of microsomal fractions
Separation of the PM fraction from mechanically damaged leaf or root tissue or suspension cells begins with the separation of the microsomal membrane fraction containing PM. The plasma membrane is then separated from the microsomal fraction by two-phase separation. All steps are performed at 4°C.
1 ) Arabidopsis leaves and roots
( 1 ) Collect leaves and roots quickly, place them on moist paper placed on ice, and rinse briefly with ice-cold distilled water. Weigh the fresh weight of the material. The ratio of homogenate to tissue is 2:1 [ medium (ml) / fresh weight (g) ]. All of the homogenate was added to the tissue and homogenized in a Waring mixer at low speed for 10s, then centrifuged at high speed 4 times for 10s each.
( 2 ) The resulting homogenate was filtered through a nylon cloth (100 μm diameter) to remove all cell wall debris.
( 3 ) The filtered homogenate was centrifuged at a maximum of 26,000 g for 25 min to precipitate chloroplasts and mitochondria.
( 4 ) The precipitate was removed and the supernatant was filtered through two successive filter papers (63 μm and 34 μm in diameter, respectively).
( 5 ) Centrifuge the supernatant for 25 min at a maximum of 84,000 g to obtain a precipitate of microsomes from the supernatant. The resulting pellet precipitate is resuspended in a total volume of 9 ml of Pellet Buffer/20-40 g fresh weight (Table 11-2).
2 ) Arabidopsis Suspension Cells
( 1 ) Pass the cells through a glass fiber of porosity 2. Suspension cells are washed with cold wash buffer (1:1, WBSC/suspension cells) (see Section 11. 2. 3 2 ).
( 2 ) Weigh the fresh weight of the material.
( 3 ) Incubate suspended cells in homogenization solution for 10 min {2.5:1, [ medium (ml)/fresh weight (g )]}.
( 4 ) Cells were pre-homogenized in a Waring mixer at low speed for 10 s, and then thoroughly broken in a cell crusher so that the cells were pulverized under high pressure gas pressure. The cells were made to pass through a pressure tunnel of 180 μm in diameter by the applied high pressure (0.54 kbar). The cells gain high velocity during the transfer from high to low pressure. Through cavitation, the emitted cells contain bursts.
( 5 ) The cells are then homogenized by centrifugation at up to 10,000 g for 10 min.
( 6 ) The supernatant is filtered through a 100 μm diameter nylon cloth.
( 7 ) The supernatant is centrifuged at up to 50,000 g for 35 min to obtain a microsomal precipitate. The precipitate is resuspended in a total volume of 9 ml of particulate buffer/20-40 g fresh weight (see Table 11-2).
2. Two-Phase Separation of Plasma Membranes
( 1 ) The separation of plasma membranes was performed using the same protocol as that used for the separation of PM from Arabidopsis suspension cells, leaves, and roots. The two-phase separation method is affected by temperature variations, so it is best to operate in a cool room at all times.
( 2 ) 27 g of the phase system is mixed with 9 g of suspension particles (up to 40 g of fresh weight). Over-sampling will affect the accuracy of the phase separation system.
( 3 ) Shake the tube vigorously 15-20 times. Ensure that the contents of the tube are well mixed and that the dextran is well mixed with the particulate suspension.
( 4 ) The system is separated into two fractions by centrifugation at 2000 g for 10 min. PM-derived vesicles are preferentially distributed into the PEG-upper phase (upl), whereas membrane vesicles from other membranes are distributed into the lower phase (lowl, Figure 11-1).
( 5 ) To purify the PM from the upper phase, the phase was re-separated in two phases to obtain the lower phase (low2, Figure 11-1).
( 6 ) The upper and lower new layer phases were obtained by mixing 9 g of micelle buffer with 27 g of the two-phase separation system.
( 7 ) After homogenization by centrifugation at 2000 g for 10 min, two new phases, up2 and low2, were obtained. upl was mixed with low2, and lowl was mixed with up2, respectively.
( 8 ) After additional homogenization and centrifugation steps, the split phases up3, low3, up4, and low4 were obtained (Figure 11-1).
( 9 ) The upper phase up3 is collected.
( 10 ) The upper phase up4 is mixed with the lower phase low3.
( 11 ) After homogenization/centrifugation, the washed up4 is collected.
( 12 ) The upper phases up3 and up4 are diluted in 60 ml of wash buffer, (Table 11-4), followed by sedimentation centrifugation at a maximum of 85,000 g for 35 min. For leaf and root suspension cells, pelletize at a maximum of 176,000 g for 30 min.
( 13 ) Precipitated PM proteins were resuspended in wash buffer with 10 μmol/L leupeptin peptide and 5 mmol/L DTT, snap-frozen in liquid nitrogen, and allowed to stand for long periods of time at -80°C.
( 14 ) Protein yield was 1.5 to 2.5 mg per 100 g fresh weight (see Note 3).
Even with careful application of the two-phase system, complete purification is virtually impossible; therefore, it is necessary to estimate the relative contamination of the PM fraction by other membrane and soluble proteins (see Note 4 and Note 5). 
3.2 Enrichment of hydrophobic proteins
Urea-sodium hydroxide treatment of PM membranes in alkali treatment resulted in better complexation of hydrophobic proteins water channel proteins (see Remark 6 and Figure 11-2). 
( 1 ) PM protein (0.5 mg) in 15 ml of buffer A (see Table 11-5) was placed on ice for 5 min and then centrifuged at up to 100,000 g for 10 min.
( 2 ) The supernatant is discarded and the resulting precipitate is resuspended in 20 mmol/L NaOH and centrifuged at up to 100,000 g for 10 min.
( 3 ) The precipitated protein is washed with Buffer B (Table 11-6) and centrifuged at up to 100,000 g for 10 min.
( 4 ) The final precipitate is resuspended in PM Preservation Buffer (Table 11-4) for protein experiments. (See Note 3).
( 5 ) The PM protein fraction is diluted twofold by Sample Buffer SB2X (Table 11-7) and subjected to SDS-PAGE electrophoresis (Figure 11-3 and see Note 7). 
3.3 Protein Separation by Bidirectional Gel Electrophoresis
1. Protein solubilization
( 1 ) PM proteins (120 μg; all PM or PM treated with sodium urea hydroxide) were enriched at up to 100,000 g for 15 min.
( 2 ) The enriched precipitate is resuspended in 350 μl of bidirectional electrophoresis solution (Table 11-8) for 1 h with constant shaking.
( 3 ) After centrifugation at 10000 g for 10 min, the supernatant is carefully collected and adjusted to 350 μl with bidirectional electrophoresis solution (Table 11-8). 
2. 2D gel electrophoresis
( 1 ) Isoelectric focusing (IEF) experiments were performed with commercially available fixed pH gradient gel strips (linear or nonlinear pH gradients 3 to 10, 18 cm long; Amersham Pharmacia Biotech) using an IPGphor device (Amersham Pharmacia Biotech).
( 2 ) Adhesive strips were rehydrated after 350 μl of sample proteins were loaded, passively hydrated for 4 h, and then actively hydrated for 7 h at a constant voltage ( 50 V).
( 3 ) The strips were focused under the following conditions: from 0 to 300 V for 1 min; 300 V for 3 h; 300 to 3500 V for 1 h and 3500 V for 80 kVh.
( 4 ) After IEF, the tape continued to equilibrate at room temperature in an equilibration solution containing 2% DTT and 2.5% mercaptoethanol (after Chevallet et al., Note 26).
( 5 ) Two-way electrophoresis (SDS-PAGE) was performed in homogeneous 11% T gels (Protean I I, Bio-Rad).
( 6 ) Gel strips were sealed on top of the electrophoresis gel with low melting point agarose [28].
( 7 ) Electrophoresis was performed at 20 mA for 1 h, then at 40 mA for 4-5 h. The electrophoresis was carried out at 20 mA for 1 h, then at 40 mA for 4-5 h.
( 8 ) To facilitate subsequent mass spectrometry analysis, the gel is stained with silver or Caulphas G-250, respectively (Figs. 11-4 and 11-5, see Note 8).