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[Bio101] Adipocyte Subcellular Fractionation
[Bio101] 脂肪细胞亚细胞分离   

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Abstract

Materials and Reagents

  1. Fat tissue
  2. Bovine serum albumin (BSA)
  3. (-)-N6-(R-Phenyl-isopropyl)-adenosine
  4. Adenosine
  5. Sodium orthovanadate
  6. Ammonium bicarbonate
  7. Sucrose
  8. Tizma base
  9. Magnesium sulphate (MgSO4·7H2O)
  10. Potassium chloride (KCl)
  11. Sodium phosphate
  12. Sodium pyrophosphate
  13.  Monopotassium phosphate
  14. Iodoacetate
  15. EDTA
  16. EGTA
  17. Sodium fluoride
  18. Glucose
  19. Calcium Chloride
  20. HEPES (ICN Biomedicals)
  21. Glucose
  22. Protease inhibitors (Roche Diagnostics)
  23. NaCl 0,9% solution (see Recipes)
  24. Collagenase solution (see Recipes)
  25. KRHLP solution (see Recipes)
  26. KRHG solution (see Recipes)
  27. PES homogenization buffer (see Recipes)
  28. Tris-EDTA solution (see Recipes)
  29. Stock solutions (see Recipes)

Equipment

  1. Beckman centrifuges and ultracentrifuges (Beckman Coulter)
  2. Rotors: JA-21, SW-41, TLS-55 , and TLA-100 (Beckman Coulter)
  3. Probe sonicator
  4. Teflon/glas homogenizer (Thermo Fisher Scientific)
  5. Gauze bandage
  6. Beckman UltraClear tubes (Nalgene & Beckman Coulter)

Procedure

  1. 1. The fat tissue is rinsed with isotonic NaCl solution (0.9%) immediately after harvesting.
    2. The tissue is cut to small pieces and incubated with collagenase (1 ml g-1 fat) for 1 h at 37 °C.
    3. KRHLP-1% BSA buffer is added and the cells are filtered first through a single and then a double layer of gauze bandage.
    4. The cells are washed with KRHLP-1% BSA buffer until the lower phase is clear.
    5. The cells are diluted with the same buffer up to 10-15% and preincubated with PIA (1μl ml-1 cell suspension) and adenosine (0.5 μl ml-1) for 10-15 min at 37 °C. The buffer is changed to PES buffer (homogenization buffer) containing 2 mM sodium orthovanadate and protease inhibitors. Up to 20-30% cells.
    6. The cells are homogenized at RT with 5 strokes in a Teflon/glass homogenizer.
    7. The homogenate is transferred to Nalgene tubes and centrifuged for 20 min at 4 °C (JA-21, 14,000 rpm).
    From now on all steps are done at 4 °C. With cold spatula the fat on the top of the tubes is removed.
    The supernatant contains intracellular membrane vesicles (microsome fraction) and soluble proteins (cytosol fraction).
    The pellet contains in addition to the plasma membrane, mitochondria and nuclei.
    8. The supernatant is transferred to Beckman UltraClear tubes and centrifuged for 75 min (SW-41, 35,000).
    9. The super containing cytosolic proteins is transferred to 15-ml tubes and frozen. The pellet (microsome) is resuspended in 200 μl Tris-EDTA/2 mM sodium orthovanadate.
    10. The pellet is resuspended in 200 μl Tris-EDTA/2 mM sodium orthovanadate and placed carefully on 1.12 M sucrose solution (400 μl). Rinse the pellet tube with 400 μl Tris-EDTA/2 mM sodium orthovanadate and put on the sucrose solution. (600 μl of suspension can be loaded on the 400 μl of 1.12 M sucrose).
    11. The sucrose tube is centrifuged for 60 min (TLS-55, 46,000 rpm). The plasma membrane will stay at the top of the sucrose solution while the mitochondria and the nuclei will sediment.
    12. The plasma membrane band (400-600 μl) is transferred to a new tube and diluted up to 1,000 μl with Tris-EDTA/2 mM sodium orthovanadate. Vortex! 10% is saved as plasma membrane sample and the rest is for caveolae preparation.
    13. The plasma membrane fraction and the caveolae fraction are pelleted (20 min, TLA-100, 69,000 rpm). The plasma membrane sample is resuspended in 200 μl Tris-EDTA/2 mM vanadate and frozen.
    14. Caveolae sample is resuspended in 200 μl carbonate buffer (pH 11) (or 50 mM NH4HCO3 + 2 mM sodium orthovanadate) and transferred to sonication tube. Caveolae tube is rinsed with additional 200 ml and the volume in the sonication tube is adjusted to 2,000 ml.
    15. The sonication probe is cooled before and sonication is performed at 16 micron (3x 20 sec, with 60 sec intervals).
    16. The sonicated sample is diluted with 2 ml 90% sucrose and placed at the bottom of an ultracentrifuge tube containing 5-35% discontinuous sucrose gradient.
    17. The tube is centrifuged for 16-20 h (SW-41, 39,000).
    18. A light-scattering band (caveolae-enriched fraction) confide to the 5-35% sucrose interface is collected (1 ml) and diluted in Tris-EDTA + 2 mM sodium orthovanadate up to 4 ml.
    19. Caveolae are pelleted for 20 min (TLA-100, 69,000 rpm).

References

  1. Aboulaich, N., Vainonen, J. P., Stralfors, P. and Vener, A. V. (2004). Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes. Biochem J 383(Pt 2): 237-248.
  2. Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K. E. and Stralfors, P. (1999). Localization of the insulin receptor in caveolae of adipocyte plasma membrane. FASEB J 13(14): 1961-1971.

简介

材料和试剂

  1. 脂肪组织
  2. 牛血清白蛋白(BSA)
  3. ( - ) - N6-(R-苯基 - 异丙基) - 腺苷
  4. 腺苷
  5. 原钒酸钠
  6. 碳酸氢铵
  7. 蔗糖
  8. Tizma基地
  9. 硫酸镁(MgSO 4·7H 2 O)
  10. 氯化钾(KCl)
  11. 磷酸钠
  12. 焦磷酸钠
  13.  磷酸二氢钠
  14. 碘乙酸盐
  15. EDTA
  16. EGTA
  17. 氟化钠
  18. 葡萄糖
  19. 氯化钙
  20. HEPES(ICN生物医学)
  21. 葡萄糖
  22. 蛋白酶抑制剂(Roche Diagnostics)
  23. NaCl 0.9%溶液(见配方)
  24. 胶原酶溶液(参见配方)
  25. KRHLP解决方案(参见配方)
  26. KRHG解决方案(参见配方)
  27. PES均质缓冲液(参见配方)
  28. T ris-EDTA solution(见Recipes)
  29. S tock解决方案(请参阅配方)

设备

  1. Beckman离心机和超速离心机(Beckman Coulter)
  2. 转子:JA-21,SW-41,TLS-55和TLA-100(Beckman Coulter)
  3. 探头超声仪
  4. Teflon/glas匀浆器(Thermo Fisher Scientific)
  5. 纱布绷带
  6. Beckman UltraClear管(Nalgene& Beckman Coulter)

程序

  1. 1.在收获后立即用等渗NaCl溶液(0.9%)冲洗脂肪组织 2.将组织切成小块,并在37℃下用胶原酶(1ml g-1脂肪)温育1小时。
    3.加入KRHLP-1%BSA缓冲液,首先通过单层,然后双层纱布绷带过滤细胞。
    4.用KRHLP-1%BSA缓冲液洗涤细胞,直到下层澄清 5.用相同的缓冲液将细胞稀释至10-15%,并在37℃下用PIA(1μl/ml细胞悬浮液)和腺苷(0.5μl/ml)预温育10-15分钟。将缓冲液变为含有2mM原钒酸钠和蛋白酶抑制剂的PES缓冲液(匀化缓冲液)。高达20-30%的细胞。
    6.将细胞在室温下用特氟隆/玻璃匀浆器匀浆5次 7.将匀浆转移到Nalgene管中,在4℃下离心20分钟(JA-21,14,000rpm)。
    从现在起,所有步骤都在4°C完成。用冷刮刀除去管顶部的脂肪。
    上清液含有细胞内膜囊泡(微粒体部分)和可溶性蛋白质(细胞质部分) 除了质膜外,颗粒还含有线粒体和核 8.将上清液转移到Beckman UltraClear管中,离心75分钟(SW-41,35,000)。
    9.将含有超级细胞溶质的蛋白质转移到15ml管中并冷冻。将沉淀(微粒体)重悬于200μlTris-EDTA/2mM原钒酸钠中 10.将沉淀重悬于200μlTris-EDTA/2mM原钒酸钠中并小心地置于1.12M蔗糖溶液(400μl)上。用400μlTris-EDTA/2mM原钒酸钠冲洗沉淀管,并放在蔗糖溶液上。 (600μl悬浮液可以装载在400μl1.12M蔗糖上) 11.将蔗糖管离心60分钟(TLS-55,46,000rpm)。质膜将保持在蔗糖溶液的顶部,而线粒体和细胞核将沉淀 将质膜带(400-600μl)转移到新管中,并用Tris-EDTA/2mM原钒酸钠稀释至1,000μl。涡流! 10%保存为质膜样品,其余为用于小窝制备 13.将细胞膜级分和细胞膜穴样内陷部分沉淀(20分钟,TLA-100,69,000rpm)。将质膜样品重悬浮于200μlTris-EDTA/2mM钒酸盐中并冷冻 14.将窖样品重悬浮于200μl碳酸盐缓冲液(pH 11)(或50mM NH 4 HCO 3 + 2mM原钒酸钠)中,并转移至超声处理管。用另外200ml冲洗窖管,并将超声处理管中的体积调节至2000ml 超声处理探针先冷却,然后以16微米(3×20秒,间隔60秒)进行超声处理。
    16.用2ml 90%蔗糖稀释经超声处理的样品,并置于含有5-35%不连续蔗糖梯度的超速离心管的底部。 17.将管离心16-20小时(SW-41,39,000)。
    18.收集到达到5-35%蔗糖界面的光散射带(窖富集部分)(1ml),并在Tris-EDTA + 2mM原钒酸钠中稀释至4ml。
    19.将小窝囊菌沉淀20分钟(TLA-100,69,000rpm)

参考文献

  1. Aboulaich,N.,Vainonen,J.P.,Stralfors,P。和Vener,A.V。(2004)。 矢量蛋白质组学揭示了人类脂肪细胞中细胞膜穴样内陷表面的聚合酶I和转录释放因子(PTRF)的靶向,磷酸化和特异性断裂。 Biochem J 383(Pt 2):237-248
  2. Gustavsson,J.,Parpal,S.,Karlsson,M.,Ramsing,C.,Thorn,H.,Borg,M.,Lindroth,M.,Peterson,KH,Magnusson,KE和Stralfors, 。 脂肪细胞质膜的细胞膜穴样中的胰岛素受体的定位。 FASEB J 13(14):1961-1971。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Aboulaich, N. (2011). Adipocyte Subcellular Fractionation. Bio-protocol Bio101: e36. DOI: 10.21769/BioProtoc.36;
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