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Membrane Preparation, Sucrose Density Gradients and Two-phase Separation Fractionation from Five-day-old Arabidopsis seedlings
利用蔗糖梯度密度离心的方法从拟南芥幼苗中分离膜蛋白   

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Abstract

Membrane preparation has been widely used for characterization the membrane proteins. Membrane fractions can be separated by a combination of differential and density-gradient centrifugation techniques (Hodges et al., 1972; Leonard and Vanderwoude, 1976). Here we firstly describe a method to isolate total microsomal fractions including plasma membrane, intracellular vesicles, Golgi membranes, endoplasma reticulum, and tonoplast (vacuolar membrane) from 5-7 days old seedlings, which is often analyzed for auxin transporters in Arabidopsis (Leonard and Vanderwoude, 1976; Titapiwatanakun, et al., 2009; Yang et al., 2013; Blakeslee et al., 2007). After homogenization, plant debris including cell walls, chloroplasts and nucleus were removed by low speed centrifugation (8,000 x g), then total microsomal membranes were pelleted by high speed centrifugation (10,000 x g) and separated from soluble fractions. We secondly describe a method to separate microsomal fractions according to size or density in a sucrose density-gradient system by centrifugation. The linear sucrose gradient from 20%-55% (1.09-1.26 g cm-3) were used to separate membranes with different densities: tonoplast, 1.10-1.12 cm-3, Golgi membranes, 1.12-1.15 cm-3, rough endoplasmic reticulum 1.15-1.17 cm-3, thylakoids, 1.16-1.18 cm-3, plasma membrane, 1.14-1.17 g cm-3, and mitochondrial membranes, 1.18-1.20 cm-3 (Leonard and Vanderwoude, 1976; Larsson et al., 1987; Briskin and Leonard, 1980). However, the plasma membrane can also be isolated according to its outer surface properties which are very different from intracellular membrane surfaces. Thus, the right-side-out plasma membrane vesicles can be separated in an aqueous Dextran-polyethylene glycol two-phase system. The plasma membranes can be purified to > 90% in the upper phase (Larsson et al., 1987; Alexandersson et al., 2008). Two-phase systems for Arabidopsis seedlings were described in the section 3. Sucrose density gradient membrane fractionation followed by western blot is often used to analyze the distribution of certain membrane protein, while Two-phase separation is used when high purity of plasma membrane or intracellular membrane is required.

Keywords: Plant membrane preparation(植物膜的制备), Sucrose density gradients(蔗糖密度梯度), Two-phase separation(两相分离), Arabidopsis(拟南芥)

Materials and Reagents

Note: All chemicals were purchased from Sigma-Aldrich (http://www.sigmaaldrich.com/united-states.html) unless otherwise specified.

  1. Arabidopsis seedlings or mature tissue
  2. Ice
  3. Sucrose (Molecular Biology, Sigma-Aldrich, catalog number: 84097 )
  4. HEPES (Sigma-Aldrich, catalog number: H-3375 )
  5. EDTA (Research Products International, catalog number: E57020 )
  6. PVP (40,000) (Fisher Scientific, catalog number: BP431 )
  7. BSA (Sigma-Aldrich, catalog number: A-7906 )
  8. DTT (Sigma-Aldrich, catalog number: D0632 )
  9. Leupeptin (Sigma-Aldrich, catalog number: L2884 )
  10. PMSF (Sigma-Aldrich, catalog number: P-7626 )
  11. Benzamidine (Sigma-Aldrich, catalog number: B-6506 )
  12. Pepstatin A (Sigma-Aldrich, catalog number: P5318 )
  13. Aprotinin (Sigma-Aldrich, catalog number: A1153 )
  14. BTP-MES (BIS-TRIS propane, Sigma-Aldrich, catalog number: B6755 ; MES, Research Products International, catalog number: M22040 )
  15. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  16. Dextran (GE, catalog number: 17-0320-01 )
  17. Polyethylene glycol 3350 (Union Carbide Corporation, catalog number: Carbowax 3350 )
  18. EGTA (Sigma-Aldrich, catalog number: E3889 )
  19. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
  20. Grinding buffer (see Recipes)
  21. Resuspension buffer (see Recipes)
  22. Stock solutions (see Recipes)
  23. Phase mixture (see Recipes)
  24. Phase system (see Recipes)

Equipment

  1. Hermle Labnet Z383 centrifuge (Labnet International)
  2. OptimaTM-L-90K ultracentrifuge (Beckman Coulter)
  3. TLX centrifuge (TLA 100.3 rotor) (Beckman Coulter)
  4. Waring blender jar (Waring Pro model: PBB212 )
  5. Mortar and pestle
  6. SW-28 rotor
  7. Nylon SS-34 tube
  8. SW-38 tube
  9. 3.5 ml polycarbonate TLA 100.3 tube
  10. 2 ml screwcap tubes
  11. 10, 15 and 50 ml Falcon tubes
  12. Polyallomer ultracentrifuge tube (Beckman Coulter)

Procedure

  1. Membrane preparation
    1. Turn on Hermle Labnet Z383 centrifuge. Set temperature to 4 °C. Collect 3-5 g of tissue from 5-d seedlings or mature tissue. Squeeze balls of tissue dry and weigh.
      1. For large quantity samples (> 40 g), add tissue to Waring blender jar and fill jar so that no air is present. Fill a bucket with ice. Pulse the waring jar for 30 s, then incubate in ice for 5 min. Repeat 3x.
      2. For small quantity samples (< 10 g), this step can be skipped, and the seedlings can be ground in grinding buffer in a mortar and pestle (see step A2).
    2. Pour into pre-chilled motar and pestle on ice. Add a little bit of sand (acid washed, Sigma 18649).
      1. Immediately grind in a mortar and pestle for approximately 10 min.
      2. Pour through Miracloth lining a plastic power funnel set into nylon SS-34 tube. Squeeze through Miracloth to extract all of liquid homogenate. Fill tubes to 1 cm from top when standing upright.
      3. If the yield is poor, or the sample still look “unground”, it can be returned to mortar and pestle, and ground again in a small aliquot of grinding buffer.
    3. Balance the tubes and centrifuge at 8,000 x g, 4 °C for 15 min.
      1. Turn on Beckman Coulter OptimaTM-L-90K ultracentrifuge and set temperature to 4 °C.
      2. Carefully pour off supernatant from SS-34 tubes immediately without disturbing the pellet. Try not to collect any of the dark green pellet material in the supernatant fraction (the pellet is very easy to disturb).
      3. Collect the cellular debris pellet and store in liquid nitrogen until use, if desired.
    4. To obtain a microsomal pellet, centrifuge the supernatant from the 8,000 x g spin at 100,000 x g, 4 °C, for 50 min, 1 h. Store additional supernatant on ice.
      1. Use thin wall tubes in the SW-28 rotor. The entire bucket assembly must be balanced within 0.005 g. Be sure to place buckets in the correct position and verify that they have engaged the rotor properly.
      2. Pour off supernatant and store at -70 °C (if soluble proteins are needed).
      3. If you have additional supernatant (from the 8,000 x g spin) on ice, pour off enough supernatant (from the 100,000 x g spin) from the ultracentrifuge tubes to allow for refilling with extra 8,000 x g supernatant. Balance tubes with poured off 100,000 x g supernatant as needed. Repeat 50 min spin.
    5. Pour off all supernatants–save as above if desired. Wipe out inside of tubes above the pellet with a Kimwipe or paper towel. Place on ice.
    6. Re-suspend each SW-28 pellet in microsomal resuspension buffer (volume will depend on pellet size and intended use, usually 1-2 ml). Use a cut-off 1 ml pipet tip to resuspend initially. This will be difficult, as the microsomes are very stick and hydrophobic. Try not to let the pellet stick to the pipet tip or walls of the centrifuge tube.
      1. After the pellet has been fairly well resuspended (pipet up and down for 100 times), use a normal 1 ml pipet tip to “break up” the clumps a bit and resuspend the microsomes more thoroughly (pipet up and down for 100 times).
      2. Finally, use a 200 μl pipet tip to break up the clumps and completely homogenize the preparation (pipet up and down for 200 times).
      3. If a wash of the pellet is desired, transfer the resuspended pellet to 3.5 ml polycarbonate TLA 100.3 tube. Wash each SW-38 tube with a bit of additional buffer, and add this to the TLA tubes. Centrifuge at 100,000 x g, 4 °C for 30 min in the TLX centrifuge (TLA 100.3 rotor). Discard supernatant, wipe out walls of tubes, store on ice.
    7. Resuspend the microsomal pellet in resuspension buffer as in step A6. If fraction is being stored, aliquot into 500 μl–1 ml traction in 2 ml screwcap tubes. Freeze in liquid nitrogen, and store in either liquid nitrogen or -80 °C freezer until use.

  2. Sucrose density gradients
    Note: Preparation of sucrose solution: Important! Sucrose must be ultra pure.
    1. Prepare 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% (w/v) sucrose solution with 20 mM HEPES pH= 7.8, 1 mM EDTA in 10 ml tubes.
      Note: Turn the tubes on a rotator for approximately 20 min until all sucrose has dissolved.
    2. On the bottom of tube (Beckman polyallomer ultracentrifuge tube) put first the drop of 55% Suc and after that 2 ml of 50%, 45%, 40%, 35%, 30%, 25% and 20% were carefully added on the top, the interface should be seen (see Figure 1). At the top of the tube upon which to pipette the 0.5 ml sample of resuspended microsomal fraction (from membrane preparation).
      Note: Allow sucrose to run down very slowly inside of tube. Sucrose layers should be visible when they are layering progressively. Place the tube with sucrose layers at 4 °C overnight to form continuous gradients.


      Figure 1. A carton to show the interface of sucrose layers. All interfaces should be clearly seen between different concentrations. Please carefully add each concentration to obtain clear interface.

    3. Centrifuge (Optima L-90K ultracentrifuge) 100,000 x g for 12 h.
    4. Immediately after the run the tube should be removed from the rotor taking care not to disturb the layers of sucrose gradient.
    5. Collect the 0.5 ml fractions from top to bottom very carefully to Eppendorf’s tubes.

  3. Two phase separation for plasma membrane and endomembrane fractions
    Stock solutions, Phase mixture (2.7 g) and Phase system (30 g) (see Recipes)
    1. Microsomal membranes are prepared as section A above.
    2. Fresh microsomal membranes are resuspended in 1 ml of buffer (0.33 M sucrose, 3 mM KCl, and 5 mM potassium phosphate, pH 7.8, and 20 mg/ml complete protease inhibitor cocktail from Sigma-Aldrich).
    3. Add 0.9 g resuspended membrane to 2.7 g two-phase separation mixture (can be scaled up).
    4. After mixing (inverting 30 times), the phases were separated by centrifugation (swinging rotor) at 1,500 x g for 5 min.
    5. The upper plasma membrane phase was carefully removed to a new tube, and mixed and repartitioned with equal weight of fresh lower phase solution (repeat one more time). Likewise, the lower phase (endosomal membrane phase) was repartitioned twice with equal weight of the upper phase.
    6. The final upper and lower phase were diluted, 3 fold and 10 fold, respectively, with 10 mM BTP-MES, pH 7.8, 1 mM EGTA, 1 mM EDTA, 0.29 M sucrose, and 20 mg/ml complete protease inhibitor cocktail, then centrifuged at 120,000 x g for 1 h.
    7. Pellets were resuspended in 10 mM BTP-MES, pH 7.8, 1 mM EGTA, 1 mM EDTA, 0.29 M sucrose, 20 mg/ml complete protease inhibitor cocktail.

Recipes

  1. Grinding buffer


    Grinding buffer
    100 ml
    300 ml
    0.29 M Sucrose
    10 g
    30 g
    25 mM HEPES, pH =8.5
    0.6 g
    1.8 g
    20mM EDTA (disodium salt)
    0.74 g
    2.23 g
    PVP(40,000)
    0.5 g
    1 g
    0.2% BSA
    0.2 g

    pH to 8.5
    Refrigerate
    Just before use, add:
    3 mM DTT (1 M Stock)
    300 μl
    900 μl
    200 ng/ml Leupeptin (200 μg/ml stock)
    100 μl
    300 μl
    PMSF (not AP assay) (100 mM in EtOH)
    100 μl
    300 μl
    200 μM Benzamidine
    100 μl
    100 μl
    (from 200 mM each stock in EtOH)


    Pepstatin A (2 mg/ml STOCK)
    100 μl

    Aprotinin (1 mg/ml STOCK)
    10 μl
    30 μl
  2. Resuspension buffer

    Resuspension buffer
    100 ml
    10 mM BTP-MES, pH 7.8 (1 M stock)
    1 ml
    250 mM sucrose
    8.56 g
    20% glycerol
    20 g
    --No GLYCEROL for membrane used in lipid raft purification
    Just before use, add:
    200 ng/ml Leupeptin (200 μg/ml stock)
    100 μl
    PMSF (not AP assay) (1,000 mM in EtOH)
    100 μl
    200 μM benzamide/benzamidine (from 200 mM each stock in EtOH)
    100 μl
    Pepstatin A (2 mg/ml STOCK)
    100 μl
    Aprotinin (1 mg/ml STOCK)
    10 μl
  3. Stock solutions
    20% (w/w) dextran in water
    40% (w/w) polyethylene glycol 3350 in water
    Incubate at 4 °C for 2-3 days
  4. Phase mixture (2.7 g)
    1.116 g 20% dextran
    0.558 g 40% polyethylene glycol 3350
    0.305 g sucrose
    67.5 μl 0.2 M potassium phosphate (pH 7.8)
    4.1 μl 2 M KCl
    Add water to a final weight of 2.7 g
  5. Phase system (30 g)
    9.3 g 20% dextran
    4.65 g 40% polyethylene glycol 3350
    3.389 g sucrose
    0.75 ml 0.2 M potassium phosphate (pH 7.8)
    45 μl 2 M KCl
    Add water to a final weight of 30 g
    The phase system is mixed and allowed to settle overnight at 4 °C
    The upper phase solution and the lower phase solution (for repartition) are collected
    Stored separately at 4 °C

Acknowledgments

The work was supported by the Department of Energy, Basic Energy Sciences, grant no. DE-FG02-06ER15804 to ASM. HY was supported as part of the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Award Number DE-SC0000997.

References

  1. Alexandersson, E., Gustavsson, N., Bernfur, K., Karlsson, A., Kjellbom, P. and Larsson, C. (2008). Purification and proteomic analysis of plant plasma membranes. Methods Mol Biol 432: 161-173. 
  2. Blakeslee, J. J., Bandyopadhyay, A., Lee, O. R., Mravec, J., Titapiwatanakun, B., Sauer, M., Makam, S. N., Cheng, Y., Bouchard, R., Adamec, J., Geisler, M., Nagashima, A., Sakai, T., Martinoia, E., Friml, J., Peer, W. A. and Murphy, A. S. (2007). Interactions among PIN-FORMED and P-glycoprotein auxin transporters in Arabidopsis. Plant Cell 19(1): 131-147. 
  3. Briskin, D. P. and Leonard, R. T. (1980). Isolation of tonoplast vesicles from tobacco protoplasts. Plant Physiol 66(4): 684-687.
  4. Hodges, T. K., Leonard, R. T., Bracker, C. E. and Keenan, T. W. (1972). Purification of an ion-stimulated adenosine triphosphatase from plant roots: association with plasma membranes. Proc Natl Acad Sci U S A 69(11): 3307-3311.
  5. Larsson, C., Widell, S. and Kjellbom, P. (1987). Preparation of high-purity plasma membranes. Methods Enzymol 148: 558-568.
  6. Leonard, R. T. and Vanderwoude, W. J. (1976). Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll. Plant Physiol 57(1): 105-114.
  7. Titapiwatanakun, B., Blakeslee, J. J., Bandyopadhyay, A., Yang, H., Mravec, J., Sauer, M., Cheng, Y., Adamec, J., Nagashima, A., Geisler, M., Sakai, T., Friml, J., Peer, W. A. and Murphy, A. S. (2009). ABCB19/PGP19 stabilises PIN1 in membrane microdomains in Arabidopsis. Plant J 57(1): 27-44.
  8. Yang, H., Richter, G. L., Wang, X., Mlodzinska, E., Carraro, N., Ma, G., Jenness, M., Chao, D. Y., Peer, W. A. and Murphy, A. S. (2013). Sterols and sphingolipids differentially function in trafficking of the Arabidopsis ABCB19 auxin transporter. Plant J 74(1): 37-47. 

Materials and Reagents

Note: All chemicals were purchased from Sigma-Aldrich (http://www.sigmaaldrich.com/united-states.html) unless otherwise specified.

  1. Arabidopsis seedlings or mature tissue
  2. Ice
  3. Sucrose (Molecular Biology, Sigma-Aldrich, catalog number: 84097 )
  4. HEPES (Sigma-Aldrich, catalog number: H-3375 )
  5. EDTA (Research Products International, catalog number: E57020 )
  6. PVP (40,000) (Fisher Scientific, catalog number: BP431 )
  7. BSA (Sigma-Aldrich, catalog number: A-7906 )
  8. DTT (Sigma-Aldrich, catalog number: D0632 )
  9. Leupeptin (Sigma-Aldrich, catalog number: L2884 )
  10. PMSF (Sigma-Aldrich, catalog number: P-7626 )
  11. Benzamidine (Sigma-Aldrich, catalog number: B-6506 )
  12. Pepstatin A (Sigma-Aldrich, catalog number: P5318 )
  13. Aprotinin (Sigma-Aldrich, catalog number: A1153 )
  14. BTP-MES (BIS-TRIS propane, Sigma-Aldrich, catalog number: B6755 ; MES, Research Products International, catalog number: M22040 )
  15. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  16. Dextran (GE, catalog number: 17-0320-01 )
  17. Polyethylene glycol 3350 (Union Carbide Corporation, catalog number: Carbowax 3350 )
  18. EGTA (Sigma-Aldrich, catalog number: E3889 )
  19. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
  20. Grinding buffer (see Recipes)
  21. Resuspension buffer (see Recipes)
  22. Stock solutions (see Recipes)
  23. Phase mixture (see Recipes)
  24. Phase system (see Recipes)

Equipment

  1. Hermle Labnet Z383 centrifuge (Labnet International)
  2. OptimaTM-L-90K ultracentrifuge (Beckman Coulter)
  3. TLX centrifuge ( TLA 100.3 rotor) (Beckman Coulter)
  4. Waring blender jar (Waring Pro model: PBB212 )
  5. Mortar and pestle
  6. SW-28 rotor
  7. Nylon SS-34 tube
  8. SW-38 tube
  9. 3.5 ml polycarbonate TLA 100.3 tube
  10. 2 ml screwcap tubes
  11. 10, 15 and 50 ml Falcon tubes
  12. Polyallomer ultracentrifuge tube (Beckman Coulter)

Procedure

  1. Membrane preparation
    1. Turn on Hermle Labnet Z383 centrifuge. Set temperature to 4 °C. Collect 3-5 g of tissue from 5-d seedlings or mature tissue. Squeeze balls of tissue dry and weigh.
      1. For large quantity samples (> 40 g), add tissue to Waring blender jar and fill jar so that no air is present. Fill a bucket with ice. Pulse the waring jar for 30 s, then incubate in ice for 5 min. Repeat 3x.
      2. For small quantity samples (< 10 g), this step can be skipped, and the seedlings can be ground in grinding buffer in a mortar and pestle (see step A2).
    2. Pour into pre-chilled motar and pestle on ice. Add a little bit of sand (acid washed, Sigma 18649).
      1. Immediately grind in a mortar and pestle for approximately 10 min.
      2. Pour through Miracloth lining a plastic power funnel set into nylon SS-34 tube. Squeeze through Miracloth to extract all of liquid homogenate. Fill tubes to 1 cm from top when standing upright.
      3. If the yield is poor, or the sample still look “unground”, it can be returned to mortar and pestle, and ground again in a small aliquot of grinding buffer.
    3. Balance the tubes and centrifuge at 8,000 x g, 4 °C for 15 min.
      1. Turn on Beckman Coulter OptimaTM-L-90K ultracentrifuge and set temperature to 4 °C.
      2. Carefully pour off supernatant from SS-34 tubes immediately without disturbing the pellet. Try not to collect any of the dark green pellet material in the supernatant fraction (the pellet is very easy to disturb).
      3. Collect the cellular debris pellet and store in liquid nitrogen until use, if desired.
    4. To obtain a microsomal pellet, centrifuge the supernatant from the 8,000 x g spin at 100,000 x g, 4 °C, for 50 min, 1 h. Store additional supernatant on ice.
      1. Use thin wall tubes in the SW-28 rotor. The entire bucket assembly must be balanced within 0.005 g. Be sure to place buckets in the correct position and verify that they have engaged the rotor properly.
      2. Pour off supernatant and store at -70 °C (if soluble proteins are needed).
      3. If you have additional supernatant (from the 8,000 x g spin) on ice, pour off enough supernatant (from the 100,000 x g spin) from the ultracentrifuge tubes to allow for refilling with extra 8,000 x g supernatant. Balance tubes with poured off 100,000 x g supernatant as needed. Repeat 50 min spin.
    5. Pour off all supernatants–save as above if desired. Wipe out inside of tubes above the pellet with a Kimwipe or paper towel. Place on ice.
    6. Re-suspend each SW-28 pellet in microsomal resuspension buffer (volume will depend on pellet size and intended use, usually 1-2 ml). Use a cut-off 1 ml pipet tip to resuspend initially. This will be difficult, as the microsomes are very stick and hydrophobic. Try not to let the pellet stick to the pipet tip or walls of the centrifuge tube.
      1. After the pellet has been fairly well resuspended (pipet up and down for 100 times), use a normal 1 ml pipet tip to “break up” the clumps a bit and resuspend the microsomes more thoroughly (pipet up and down for 100 times).
      2. Finally, use a 200 μl pipet tip to break up the clumps and completely homogenize the preparation (pipet up and down for 200 times).
      3. If a wash of the pellet is desired, transfer the resuspended pellet to 3.5 ml polycarbonate TLA 100.3 tube. Wash each SW-38 tube with a bit of additional buffer, and add this to the TLA tubes. Centrifuge at 100,000 x g, 4 °C for 30 min in the TLX centrifuge (TLA 100.3 rotor). Discard supernatant, wipe out walls of tubes, store on ice.
    7. Resuspend the microsomal pellet in resuspension buffer as in step A6. If fraction is being stored, aliquot into 500 μl–1 ml traction in 2 ml screwcap tubes. Freeze in liquid nitrogen, and store in either liquid nitrogen or -80 °C freezer until use.

  2. Sucrose density gradients
    Note: Preparation of sucrose solution: Important! Sucrose must be ultra pure.
    1. Prepare 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% (w/v) sucrose solution with 20 mM HEPES pH= 7.8, 1 mM EDTA in 10 ml tubes.
      Note: Turn the tubes on a rotator for approximately 20 min until all sucrose has dissolved.
    2. On the bottom of tube (Beckman polyallomer ultracentrifuge tube) put first the drop of 55% Suc and after that 2 ml of 50%, 45%, 40%, 35%, 30%, 25% and 20% were carefully added on the top, the interface should be seen (see Figure 1). At the top of the tube upon which to pipette the 0.5 ml sample of resuspended microsomal fraction (from membrane preparation).
      Note: Allow sucrose to run down very slowly inside of tube. Sucrose layers should be visible when they are layering progressively. Place the tube with sucrose layers at 4 °C overnight to form continuous gradients.


      Figure 1. A carton to show the interface of sucrose layers. All interfaces should be clearly seen between different concentrations. Please carefully add each concentration to obtain clear interface.

    3. Centrifuge (Optima L-90K ultracentrifuge) 100,000 x g for 12 h.
    4. Immediately after the run the tube should be removed from the rotor taking care not to disturb the layers of sucrose gradient.
    5. Collect the 0.5 ml fractions from top to bottom very carefully to Eppendorf’s tubes.

  3. Two phase separation for plasma membrane and endomembrane fractions
    Stock solutions, Phase mixture (2.7 g) and Phase system (30 g) (see Recipes)
    1. Microsomal membranes are prepared as section A above.
    2. Fresh microsomal membranes are resuspended in 1 ml of buffer (0.33 M sucrose, 3 mM KCl, and 5 mM potassium phosphate, pH 7.8, and 20 mg/ml complete protease inhibitor cocktail from Sigma-Aldrich).
    3. Add 0.9 g resuspended membrane to 2.7 g two-phase separation mixture (can be scaled up).
    4. After mixing (inverting 30 times), the phases were separated by centrifugation (swinging rotor) at 1,500 x g for 5 min.
    5. The upper plasma membrane phase was carefully removed to a new tube, and mixed and repartitioned with equal weight of fresh lower phase solution (repeat one more time). Likewise, the lower phase (endosomal membrane phase) was repartitioned twice with equal weight of the upper phase.
    6. The final upper and lower phase were diluted, 3 fold and 10 fold, respectively, with 10 mM BTP-MES, pH 7.8, 1 mM EGTA, 1 mM EDTA, 0.29 M sucrose, and 20 mg/ml complete protease inhibitor cocktail, then centrifuged at 120,000 x g for 1 h.
    7. Pellets were resuspended in 10 mM BTP-MES, pH 7.8, 1 mM EGTA, 1 mM EDTA, 0.29 M sucrose, 20 mg/ml complete protease inhibitor cocktail.

Recipes

  1. Grinding buffer


    Grinding buffer
    100 ml
    300 ml
    0.29 M Sucrose
    10 g
    30 g
    25 mM HEPES, pH =8.5
    0.6 g
    1.8 g
    20mM EDTA (disodium salt)
    0.74 g
    2.23 g
    PVP(40,000)
    0.5 g
    1 g
    0.2% BSA
    0.2 g

    pH to 8.5
    Refrigerate
    Just before use, add:
    3 mM DTT (1 M Stock)
    300 μl
    900 μl
    200 ng/ml Leupeptin (200 μg/ml stock)
    100 μl
    300 μl
    PMSF (not AP assay) (100 mM in EtOH)
    100 μl
    300 μl
    200 μM Benzamidine
    100 μl
    100 μl
    (from 200 mM each stock in EtOH)


    Pepstatin A (2 mg/ml STOCK)
    100 μl

    Aprotinin (1 mg/ml STOCK)
    10 μl
    30 μl
  2. Resuspension buffer

    Resuspension buffer
    100 ml
    10 mM BTP-MES, pH 7.8 (1 M stock)
    1 ml
    250 mM sucrose
    8.56 g
    20% glycerol
    20 g
    --No GLYCEROL for membrane used in lipid raft purification
    Just before use, add:
    200 ng/ml Leupeptin (200 μg/ml stock)
    100 μl
    PMSF (not AP assay) (1,000 mM in EtOH)
    100 μl
    200 μM benzamide/benzamidine (from 200 mM each stock in EtOH)
    100 μl
    Pepstatin A (2 mg/ml STOCK)
    100 μl
    Aprotinin (1 mg/ml STOCK)
    10 μl
  3. Stock solutions
    20% (w/w) dextran in water
    40% (w/w) polyethylene glycol 3350 in water
    Incubate at 4 °C for 2-3 days
  4. Phase mixture (2.7 g)
    1.116 g 20% dextran
    0.558 g 40% polyethylene glycol 3350
    0.305 g sucrose
    67.5 μl 0.2 M potassium phosphate (pH 7.8)
    4.1 μl 2 M KCl
    Add water to a final weight of 2.7 g
  5. Phase system (30 g)
    9.3 g 20% dextran
    4.65 g 40% polyethylene glycol 3350
    3.389 g sucrose
    0.75 ml 0.2 M potassium phosphate (pH 7.8)
    45 μl 2 M KCl
    Add water to a final weight of 30 g
    The phase system is mixed and allowed to settle overnight at 4 °C
    The upper phase solution and the lower phase solution (for repartition) are collected
    Stored separately at 4 °C

Acknowledgments

The work was supported by the Department of Energy, Basic Energy Sciences, grant no. DE-FG02-06ER15804 to ASM. HY was supported as part of the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Award Number DE-SC0000997.

References

  1. Alexandersson, E., Gustavsson, N., Bernfur, K., Karlsson, A., Kjellbom, P. and Larsson, C. (2008). Purification and proteomic analysis of plant plasma membranes. Methods Mol Biol 432: 161-173. 
  2. Blakeslee, J. J., Bandyopadhyay, A., Lee, O. R., Mravec, J., Titapiwatanakun, B., Sauer, M., Makam, S. N., Cheng, Y., Bouchard, R., Adamec, J., Geisler, M., Nagashima, A., Sakai, T., Martinoia, E., Friml, J., Peer, W. A. and Murphy, A. S. (2007). Interactions among PIN-FORMED and P-glycoprotein auxin transporters in Arabidopsis. Plant Cell 19(1): 131-147. 
  3. Briskin, D. P. and Leonard, R. T. (1980). Isolation of tonoplast vesicles from tobacco protoplasts. Plant Physiol 66(4): 684-687.
  4. Hodges, T. K., Leonard, R. T., Bracker, C. E. and Keenan, T. W. (1972). Purification of an ion-stimulated adenosine triphosphatase from plant roots: association with plasma membranes. Proc Natl Acad Sci U S A 69(11): 3307-3311.
  5. Larsson, C., Widell, S. and Kjellbom, P. (1987). Preparation of high-purity plasma membranes. Methods Enzymol 148: 558-568.
  6. Leonard, R. T. and Vanderwoude, W. J. (1976). Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll. Plant Physiol 57(1): 105-114.
  7. Titapiwatanakun, B., Blakeslee, J. J., Bandyopadhyay, A., Yang, H., Mravec, J., Sauer, M., Cheng, Y., Adamec, J., Nagashima, A., Geisler, M., Sakai, T., Friml, J., Peer, W. A. and Murphy, A. S. (2009). ABCB19/PGP19 stabilises PIN1 in membrane microdomains in Arabidopsis. Plant J 57(1): 27-44.
  8. Yang, H., Richter, G. L., Wang, X., Mlodzinska, E., Carraro, N., Ma, G., Jenness, M., Chao, D. Y., Peer, W. A. and Murphy, A. S. (2013). Sterols and sphingolipids differentially function in trafficking of the Arabidopsis ABCB19 auxin transporter. Plant J 74(1): 37-47. 

简介

膜制备已广泛用于表征膜蛋白。膜级分可以通过差示和密度梯度离心技术的组合来分离(Hodges等人,1972; Leonard和Vanderwoude,1976)。在这里我们首先描述一种从5-7天龄幼苗中分离总微粒体级分包括质膜,胞内囊泡,高尔基体膜,内质网和液泡膜(液泡膜)的方法,其通常在拟南芥中分析生长素转运蛋白(Leonard和Vanderwoude,1976; Titapiwatanakun,,2009; Yang等人,2013; Blakeslee等人 >,2007)。均化后,通过低速离心(8,000×g)除去包括细胞壁,叶绿体和细胞核的植物碎片,然后通过高速离心(10,000×g/ml)沉淀总微粒体膜, )并与可溶性级分分离。我们第二次描述了通过离心在蔗糖密度梯度系统中根据大小或密度分离微粒体部分的方法。使用20%-55%(1.09-1.26g cm -3)的线性蔗糖梯度分离具有不同密度的膜:tonoplast,1.10-1.12cm 3 - ,高尔基体膜,1.12-1.15cm 3,粗糙的内质网1.15-1.17cm 3,类囊体,1.16-1.18cm 3 - ,质膜,1.14-1.17g cm -3 - 和线粒体膜,1.18-1.20cm -3(Leonard和Vanderwoude,1976; Larsson等人, ,1987; Briskin and Leonard,1980)。然而,质膜也可根据其与细胞内膜表面非常不同的外表面性质分离。因此,右侧出来的质膜囊泡可以在葡聚糖 - 聚乙二醇水相两相体系中分离。可以在上层相中将质膜纯化至> 90%(Larsson等人,1987; Alexandersson等人,2008)。用于拟南芥幼苗的两相系统在第3节中描述。​​蔗糖密度梯度膜分级分离和随后的蛋白质印迹常常用于分析某些膜蛋白的分布,而使用两相分离需要高纯度的质膜或细胞内膜。

关键字:植物膜的制备, 蔗糖密度梯度, 两相分离, 拟南芥

材料和试剂

注意:所有化学品均购自Sigma-Aldrich( http://www.sigmaaldrich .com/united-states.html ),除非另有说明。

  1. 拟南芥幼苗或成熟组织
  2. 冰块
  3. 蔗糖(分子生物学,Sigma-Aldrich,目录号:84097)
  4. HEPES(Sigma-Aldrich,目录号:H-3375)
  5. EDTA(Research Products International,目录号:E57020)
  6. PVP(40,000)(Fisher Scientific,目录号:BP431)
  7. BSA(Sigma-Aldrich,目录号:A-7906)
  8. DTT(Sigma-Aldrich,目录号:D0632)
  9. 亮肽素(Sigma-Aldrich,目录号:L2884)
  10. PMSF(Sigma-Aldrich,目录号:P-7626)
  11. 苄脒(Sigma-Aldrich,目录号:B-6506)
  12. 胃酶抑素A(Sigma-Aldrich,目录号:P5318)
  13. 抑肽酶(Sigma-Aldrich,目录号:A1153)
  14. BTP-MES(BIS-TRIS丙烷,Sigma-Aldrich,目录号:B6755; MES,Research Products International,目录号:M22040)
  15. 甘油(Sigma-Aldrich,目录号:G5516)
  16. 葡聚糖(GE,目录号:17-0320-01)
  17. 聚乙二醇3350(Union Carbide Corporation,目录号:Carbowax 3350)
  18. EGTA(Sigma-Aldrich,目录号:E3889)
  19. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)
  20. 研磨缓冲器(参见配方)
  21. 重悬缓冲液(见配方)
  22. 库存解决方案(参见配方)
  23. 相混合物(参见配方)
  24. 阶段系统(参见配方)

设备

  1. Hermle Labnet Z383离心机(Labnet International)
  2. Optima TM sup-L-90K超速离心机(Beckman Coulter)
  3. TLX离心机(TLA 100.3转子)(Beckman Coulter)
  4. Waring搅拌机瓶(Waring Pro型号:PBB212)
  5. 砂浆和杵
  6. SW-28转子
  7. 尼龙SS-34管
  8. SW-38管
  9. 3.5ml聚碳酸酯TLA 100.3管
  10. 2 ml螺丝帽管
  11. 10,15和50ml Falcon管
  12. 聚合物超速离心管(Beckman Coulter)

程序

  1. 膜制备
    1. 打开Hermle Labnet Z383离心机。 将温度设置为4°C。 从5-d幼苗或成熟组织收集3-5g组织。 挤压组织球干并称重。
      1. 对于大量样品(> 40g),将组织添加到Waring混合器罐和填充罐中,使得不存在空气。 在冰桶里装冰。 脉冲维持罐30秒,然后在冰中孵育 5分钟。 重复3x。
      2. 对于少量样品(<10g),可以跳过该步骤,并且可以在研钵和杵中研磨缓冲液中研磨秧苗(参见步骤A2)。
    2. 倒入预冷的蛾和杵在冰上。 加入一点沙子(酸洗,Sigma 18649)。
      1. 立即用研钵和杵研磨约10分钟。
      2. 通过Miracloth衬里塑料功率漏斗设置到尼龙SS-34管。 挤压通过Miracloth提取所有的液体匀浆。 直立时从顶部填充管至1厘米。
      3. 如果产量差,或者样品仍然看起来"未研磨",它可以返回到研钵和研杵,并再次在一小部分研磨缓冲液中研磨。
    3. 平衡试管并在8,000×g,4℃离心15分钟。
      1. 打开Beckman Coulter Optima TM -L-90K超速离心机并将温度设置为4℃。
      2. 小心地从SS-34管上清除上清液,而不打扰沉淀。 尝试不收集任何在上清液部分的深绿色沉淀物质(沉淀是非常容易打扰)。
      3. 收集细胞碎片沉淀,并储存在液氮中,直到使用,如果需要
    4. 为了获得微粒体沉淀,将来自8000×g旋转的上清液在100,000×g,4℃下离心50分钟,1小时。将额外的上清液储存在冰上。
      1. 在SW-28转子中使用薄壁管。整个铲斗组件必须在0.005 g内平衡。确保将铲斗放置在正确的位置,并确认它们已正确接合转子。
      2. 倒出上清液并储存在-70°C(如果需要可溶性蛋白质)。
      3. 如果在冰上具有另外的上清液(来自8,000xg转速),从超速离心管倒出足够的上清液(来自100,000xg旋转),以允许用额外的8,000×g 上清液。平衡管,根据需要倒出100,000×g的上清液。重复50分钟旋转。
    5. 倒出所有上清液 - 如上所述,如果需要。用Kimwipe或纸巾擦拭颗粒上方的管子内部。放在冰上。
    6. 重悬在微粒体重悬浮缓冲液(体积将取决于丸大小和预期用途,通常1-2毫升)每个SW-28丸。使用截止1毫升移液器吸头重新悬浮起初。这将是困难的,因为微粒体是非常粘和疏水的。尝试不要让丸粒粘在移液管尖端或离心管的壁上。
      1. 在沉淀已经相当好地重悬(移液器上下100次)后,使用正常的1ml移液管尖端以"破碎"凝块,并更彻底地重悬微粒体(移液器上下移动100次)。
      2. 最后,使用200μl移液管尖端打碎块,并完全均匀的准备(移液器上下200次)。
      3. 如果需要洗涤沉淀,将重新悬浮的沉淀转移到3.5ml聚碳酸酯TLA 100.3管中。用一点额外的缓冲液洗涤每个SW-38管,并将其添加到TLA管。在TLX离心机(TLA 100.3转子)中在100,000×g离心30分钟,4℃。弃去上清液,擦干管壁,存放在冰上
    7. 如步骤A6中那样将微粒体沉淀重悬在重悬浮缓冲液中。如果级分被储存,等分到500μl-1ml牵引在2ml螺旋盖管。在液氮中冷冻,并储存在液氮或-80°C冰箱直到使用
  2. 蔗糖密度梯度
    注意:蔗糖溶液的制备:重要!蔗糖必须是超纯的。<​​/em>
    1. 在10ml试管中制备20%,25%,30%,35%,40%,45%,50%,55%(w/v)蔗糖溶液与20mM HEPES pH = 7.8,1mM EDTA。 > 注意:在旋转器上旋转管约20分钟,直到所有蔗糖溶解。
    2. 在管(Beckman polyallomer超速离心管)的底部首先放入55%Suc的滴,然后将2ml的50%,45%,40%,35%,30%,25%和20%顶部,应该看到界面(见图1)。在移取0.5ml重悬的微粒体部分样品的管的顶部(来自膜制备)。
      注意:让蔗糖在管内非常缓慢地流下。蔗糖层在它们逐渐分层时应该是可见的。将具有蔗糖层的管在4℃下放置过夜以形成连续梯度。


      图1.显示蔗糖层界面的纸箱。应该清楚地看到不同浓度之间的所有界面。请仔细添加各浓度,以获得清晰的界面。

    3. 离心机(Optima L-90K超速离心机)100,000×g 12小时。
    4. 运行后立即从转子上取下管子,注意不要打扰蔗糖梯度层
    5. 从上到下非常小心地将0.5ml级分收集到Eppendorf管中
  3. 质膜和内膜部分的两相分离
    储备溶液,相混合物(2.7g)和相系统(30g)(参见配方)
    1. 微粒体膜如上述A节所述制备
    2. 将新鲜微粒体膜重悬于1ml缓冲液(0.33M蔗糖,3mM KCl和5mM磷酸钾,pH 7.8和20mg/ml来自Sigma-Aldrich的完全蛋白酶抑制剂混合物)中。
    3. 将0.9g重悬浮的膜加入到2.7g两相分离混合物中(可以放大)。
    4. 混合(倒置30次)后,通过在1,500×g离心(摇摆转子)5分钟分离各相。
    5. 将上层细胞膜相小心地移至新管,并与等重量的新鲜下层溶液混合并重新分配(重复一次)。同样,下相(内体膜相)用等重量的上层相重新分配两次。
    6. 最终的上和下相用10mM BTP-MES,pH7.8,1mM EGTA,1mM EDTA,0.29M蔗糖和20mg/ml完全蛋白酶抑制剂混合物分别稀释3倍和10倍,然后在120,000×g离心1小时。
    7. 将沉淀重悬于10mM BTP-MES,pH7.8,1mM EGTA,1mM EDTA,0.29M蔗糖,20mg/ml完全蛋白酶抑制剂混合物中。

食谱

  1. 研磨缓冲器


    研磨缓冲器
    100 ml
    300 ml
    0.29 M蔗糖
    10克
    30克
    25mM HEPES,pH = 8.5 0.6 g
    1.8 g
    20mM EDTA(二钠盐) 0.74克
    2.23克
    PVP(40,000)
    0.5克
    1克
    0.2%BSA 0.2 g

    pH至8.5
    冷藏
    使用前,请添加:
    3 mM DTT(1 M股票)
    300微升
    900μl
    200ng/ml亮肽素(200μg/ml母液)
    100微升
    300微升
    PMSF(非AP测定)(100mM,在EtOH中) 100微升
    300微升
    200μM苄脒
    100微升
    100微升
    (从200mM每种在EtOH中的储备液)

    胃酶抑素A(2mg/ml STOCK)
    100微升

    抑肽酶(1mg/ml STOCK)
    10微升
    30微升
  2. 重悬缓冲液

    重悬缓冲液
    100 ml
    10mM BTP-MES,pH7.8(1M储备液) 1 ml
    250mM蔗糖 8.56克
    20%甘油 20克
    - 用于脂筏净化的膜的无GLYCEROL
    使用前,请添加:
    200ng/ml亮肽素(200μg/ml母液)
    100微升
    PMSF(不是AP测定)(在EtOH中为1,000mM) 100微升
    200μM苯甲酰胺/苯甲脒(从200mM每种在EtOH中的储备液) 100微升
    胃酶抑素A(2mg/ml STOCK)
    100微升
    抑肽酶(1mg/ml STOCK)
    10微升
  3. 库存解决方案
    20%(w/w)葡聚糖水溶液 40%(w/w)聚乙二醇3350的水溶液 在4℃孵育2-3天
  4. 相混合物(2.7g) 1.116g 20%葡聚糖
    0.558g 40%聚乙二醇3350
    0.305克蔗糖 67.5μl0.2M磷酸钾(pH7.8)
    4.1μl2 M KCl
    加水至最终重量为2.7克
  5. 相系统(30g)
    9.3g 20%葡聚糖 4.65g 40%聚乙二醇3350
    3.389克蔗糖 0.75ml 0.2M磷酸钾(pH7.8) 45μl2 M KCl
    加水至最终重量为30克
    将相体系混合并使其在4℃下沉降过夜 收集上相溶液和下相溶液(用于重新分配) 在4°C单独存储

致谢

这项工作得到了能源部,基础能源科学部的资助。 DE-FG02-06ER15804至ASM。 HY作为由美国能源部,科学办公室,基础能源科学办公室,奖号DE-SC0000997资助的能源前沿研究中心的生物质直接催化转化生物质(C3Bio)中心的一部分提供支持。

参考文献

  1. Alexandersson,E.,Gustavsson,N.,Bernfur,K.,Karlsson,A.,Kjellbom,P.and Larsson,C。(2008)。 植物质膜的纯化和蛋白质组学分析。 em> 432:161-173。 
  2. Blakeslee,JJ,Bandyopadhyay,A.,Lee,OR,Mravec,J.,Titapiwatanakun,B.,Sauer,M.,Makam,SN,Cheng,Y.,Bouchard,R.,Adamec,J.,Geisler,M 。,Nagashima,A.,Sakai,T.,Martinoia,E.,Friml,J.,Peer,WA和Murphy,AS(2007)。 拟南芥中PIN-FORMED和P-糖蛋白生长素转运蛋白之间的相互作用。 植物细胞 19(1):131-147。 
  3. Briskin,D.P。和Leonard,R.T。(1980)。 从烟草原生质体中分离叶绿体泡囊。 植物生理学 66(4):684-687
  4. Hodges,T.K.,Leonard,R.T.,Bracker,C.E.and Keenan,T.W。(1972)。 从植物根中纯化离子刺激的腺苷三磷酸酶:与质膜结合。 Proc Natl Acad Sci USA 69(11):3307-3311。
  5. Larsson,C.,Widell,S。和Kjellbom,P。(1987)。 高纯度质膜的制备方法Enzymol 148:558-568。
  6. Leonard,R.T.and Vanderwoude,W.J。(1976)。 通过蔗糖密度梯度离心从玉米根中分离质膜:ficoll的异常效应。 a> Plant Physiol 57(1):105-114
  7. 这些研究结果表明,ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ai ,T.,Friml,J.,Peer,WA和Murphy,AS(2009)。 ABCB19/PGP19可稳定拟南芥中的膜微区中的PIN1。 > Plant J 57(1):27-44
  8. Yang,H.,Richter,G.L.,Wang,X.,Mlodzinska,E.,Carraro,N.,Ma,G.,Jenness,M.,Chao,D.Y.,Peer,W.A.and Murphy, 固醇和鞘脂在运输拟南芥ABCB19生长素转运蛋白方面有差异功能。 植物J 74(1):37-47。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yang, H. and Murphy, A. (2013). Membrane Preparation, Sucrose Density Gradients and Two-phase Separation Fractionation from Five-day-old Arabidopsis seedlings. Bio-protocol 3(24): e1014. DOI: 10.21769/BioProtoc.1014.
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