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[Bio101] Large Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast
[Bio101] 大规模非变性亲和层析技术纯化酵母的可溶膜蛋白   

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Abstract

This protocol can be used to purify membrane proteins from yeast samples under native conditions at a large scale. This protocol has been developed primarily for FLAG-tagged proteins. This protocol can however be slightly modified and applied to other tags, such as GST or HA.

Materials and Reagents

  1. EDTA free protease inhibitors (Roche Diagnostics)
  2. Digitonin (EMD Chemicals)
  3. Protease Inhibitors (DMSO, leupeptin, pepstatin) (Sigma-Aldrich)
  4. ANTI-Flag M2 affinity gel (Sigma-Aldrich)
  5. 3x FLAG peptide (Sigma-Aldrich)
  6. NaF# (Ser/Thr phosphatase inhibitor)
  7. Na3VO4 (Tyr phosphatase inhibitor)
  8. HEPES
  9. KOAc
  10. Mg(OAc)2
  11. CaCl2
  12. Sorbitol
  13. Glycerol
  14. KOH
  15. PMSF
  16. DMSO
  17. Lysis buffer (see Recipes)
  18. Immunoprecipitation buffer (see Recipes)
  19. 50 ml lysis buffer (see Recipes) 
  20. 14 ml IP buffer with 2% digitonin (see Recipes)
  21. 20 ml IP buffer with 0.1% digitonin (see Recipes)
  22. 40 ml IP buffer with 0.1% digitonin (see Recipes)
  23. 3x FLAG elution buffer (see Recipes)

Equipment

  1. Avestin Emulsiflex C3 homogenizer (Avestin®)
  2. Beckman centrifuge and Type 70 Ti rotor (Beckman Coulter) 
  3. Beckman polycarbonate centrifuge tubes (Beckman Coulter, catalog number: 355631 )
  4. 1 L centrifuge bottles
  5. 50 ml conical tubes

Procedure

  1. Preparation of cell lysate
    1. Collect 4,000 OD of cells collected at OD600~1.4. Split to 4x 1 L centrifuge bottles. Spin at 5,000 rpm for 20 min using Rotor JLA 8.100.
    2. Wash samples in each bottle with 100 ml H2O. Transfer to 50 ml conical tubes. Store at -80 °C if necessary.
    3. Resuspend cells in 50 ml ice-cold lysis buffer supplemented with protease inhibitors (no DTT) and phosphatase inhibitors (add 50 ml buffer to first bottle, vortex, transfer all to the next bottle and so on. Eventually you will have two 50 ml conical tubes of sample).
    4. Use Avestin Emulsiflex C3 homogenizer to disrupt cells.
    5. Clear unlysed cells by centrifugation at 1,000 x g twice or three times.
    6. Transfer supernatant to Beckman polycarbonate tubes (the filling level is 16.5 ml in Type 70 Ti rotor). Use a Type 70 Ti rotor for high-speed spin at 44,000 x g (24,453 rpm) for 30 min to pellet microsomes. 
    7. Microsomes are resuspended in 0.5 ml lysis buffer and then diluted with 14 ml immunoprecipitation buffer with 2% digitonin supplemented with protease inhibitors.
    8. Membrane proteins can be solubilized by nutating lysate at 4 °C for 1.5 h. Remove unsolubilized material by centrifugation at 44,000 x g for 30 min (24,453 rpm in a Type 70 Ti rotor).

  2. FLAG Fusion Protein Immunoprecipitation
    1. Use 300 μl of gel suspension per reaction (~150 μl of packed gel volume).
    2. Thoroughly suspend the ANTI-FLAG M2 affinity gel in the vial and transfer them to a 15 ml conical tube. 
    3. Centrifuge the resin for 1 min at 400 x g. Wait for 1-2 min before handling the samples. Remove the supernatant with a gel-loading pipette tip.
    4. Wash the packed gel 4x with 5 ml of immunoprecipitation buffer with 0.1% digitonin. 
    5. Add the ~15ml cell lysate to the ANTI-FLAG resin. Immuoprecipitations are rotated for 3 h or overnight at 4 °C.
    6. Wash the resin 4x with 10 ml immunoprecipitation buffer with 0.1% digitonin. Transfer the beads into an Eppendorf tube.

  3. Elution of the FLAG –fusion protein
    Elution with 3x FLAG peptide. This elution efficiency is very high using this method.
    1. Prepare 3x FLAG Elution Buffer. Add 150 μl of 1x immunoprecipitation buffer with 0.25% digitonin and 1 μg/μl 3x FLAG peptide.
    2. Add 150 μl of 3x FLAG elution buffer to the resin in the test tube.
    3. Incubate the samples with gentle shaking for 30 min at 4 °C.
    4. Centrifuge the resin for 1 min at 400~1000 x g. Transfer the supernatant to fresh test tubes. 
    5. Repeat eluation with another 150 μl of 3x FLAG elution buffer with 0.25% digitonin and 0.1 μg/μl 3x FLAG peptide. Combine the eluates.

Recipes

  1. Lysis buffer
    Lysis buffer
    1 L
    2x  buffer without detergent
    50 mM HEPES (FW 238.3)

    23.83 g
    50 mM KOAc* (FW 98.14)

    9.814 g
    2 mM Mg(OAc)2

    4 ml of 1 M Mg(OAc)2
    1 mM CaCl2

    20 ml of 0.1 M CaCl2
    200 mM sorbitol (FW 182.17)

    72.87 g
    1 mM NaF# (Ser/Thr phosphatase inhibitor)

    0.084 g
    0.3 mM Na3VO4 (Tyr phosphatase inhibitor)

    0.1103 g

  2. Immunoprecipitation buffer
    Immunoprecipitation buffer
    1 L
    2x buffer without detergent
    50 mM HEPES-KOH (pH 6.8)

    23.83 g
    50 mM KOAc* (FW 98.14)

    9.814 g
    2 mM Mg(OAc)2

    4 ml of 1 M Mg(OAc)2
    1 mM CaCl2

    20 ml of 0.1 M CaCl2
    15% glycerol (v/v)

    300 ml of 100% glycerol
    Add Mili-Q H2O to final volume, adjust to pH=6.8 using 0.5 M KOH.
    Add the following protease inhibitors per 50 ml immunoprecipitation buffer before use: 1 complete tablet, EDTA free protease inhibitors (crush tablet first in weigh paper).
    Add 100 μl of 0.5 M PMSF in DMSO.
  3. 50 ml lysis buffer
    25 ml 2x lysis buffer
    25 ml H2O
    1 EDTA free protease inhibitor tablet
    100 μl of 0.5 M PMSF in DMSO
  4. 14 ml of IP buffer with 2% digitonin
    7 ml of 2x IP buffer
    2.8 ml of 10% digitonin
    1ml EDTA free protease inhibitor tablet
    28 μl of 0.5 M PMSF in DMSO
    4.2 ml of H2O
  5. 20 ml of IP buffer with 0.1% digitonin
    10 ml of 2x IP buffer
    0.2 ml of 10% digitonin
    9.8 ml of H2O
    Protease inhibitors (20 μl of PMSF, leupeptin, and pepstatin)
  6. 40 ml of IP buffer with 0.1% digitonin
    20 ml of 2x IP buffer
    0.4 ml of 10% digitonin
    19.6 ml of H2O
    Protease inhibitors (40 μl of PMSF, leupeptin, and pepstatin)
  7. 150 μl of elution buffer with 1μg/μl 3x FLAG peptide
    75 μl of 2x IP buffer
    3.75 μl of 10% digitonin
    30 μl of 5 mg/ml 3x FLAG peptide
    41.25 μl of H2O
  8. To make 150 μl of elution buffer with 0.1 μg/μl 3x FLAG peptide
    75 μl of 2x IP buffer
    3.75 μl of 10% digitonin
    3 μl of 5 mg/ml 3x FLAG peptide
    68.25 μl of H2O

Acknowledgments

This protocol has been modified and adapted in the Espenshade Lab, Johns Hopkins School of Medicine. Funding to support different projects that have used this protocol has come from NIH – National Heart, Lung, and Blood Institute, National Institute of Allergy and Infectious Diseases, the Pancreatic Cancer Action Network, and the American Heart Association.

References

  1. Breslow, D. K., Collins, S. R., Bodenmiller, B., Aebersold, R., Simons, K., Shevchenko, A., Ejsing, C. S. and Weissman, J. S. (2010). Orm family proteins mediate sphingolipid homeostasis. Nature 463(7284): 1048-1053.

简介

该方案可用于在天然条件下大规模地从酵母样品中纯化膜蛋白。 该协议主要开发用于FLAG标记的蛋白质。 然而,此协议可以稍微修改并应用于其他标签,如GST或HA。

材料和试剂

  1. EDTA游离蛋白酶抑制剂(Roche Diagnostics)
  2. digitonin(EMD Chemicals)
  3. 蛋白酶抑制剂(DMSO,亮抑酶肽,胃蛋白酶抑制剂)(Sigma-Aldrich)
  4. 将抗Flag M2亲和凝胶(Sigma-Aldrich)
  5. 3x FLAG肽(Sigma-Aldrich)
  6. NaF#(Ser/Thr磷酸酶抑制剂)
  7. Na3VO4(Tyr磷酸酶抑制剂)
  8. HEPES
  9. KOAc
  10. Mg(OAc)2
  11. CaCl <2>
  12. 山梨醇
  13. 甘油
  14. KOH
  15. PMSF
  16. DMSO
  17. 裂解缓冲液(见配方)
  18. 免疫沉淀缓冲液(见配方)
  19. 50ml裂解缓冲液(见Recipes)
  20. 14ml含2%洋地黄皂苷的IP缓冲液(见配方)
  21. 20ml具有0.1%洋地黄皂苷的IP缓冲液(参见Recipes)
  22. 40ml具有0.1%洋地黄皂苷的IP缓冲液(参见Recipes)
  23. 3x FLAG洗脱缓冲液(参见配方)

设备

  1. Avestin Emulsiflex C3匀浆器(Avestin )
  2. Beckman离心机和70型Ti转子(Beckman Coulter)
  3. Beckman聚碳酸酯离心管(Beckman Coulter,目录号:355631)
  4. 1升离心瓶
  5. 50ml锥形管

程序

  1. 细胞裂解液的制备
    1. 收集在OD 600〜1.4收集的4,000个OD的细胞。 分装到4x 1L离心瓶中。 使用Rotor JLA 8.100在5,000rpm旋转20分钟。
    2. 用100ml H 2 O洗涤每个瓶中的样品。 转移到50ml锥形管。 如有必要,储存于-80°C。
    3. 重悬细胞在50毫升冰冷的裂解缓冲液补充蛋白酶抑制剂(无DTT)和磷酸酶抑制剂(加入50毫升缓冲液到第一瓶,涡旋,转移到下一个瓶子,最后你会有两个50毫升锥形 样品管)。
    4. 使用Avestin Emulsiflex C3匀浆器破碎细胞。
    5. 通过在1,000×g下离心两次或三次清除未溶解的细胞。
    6. 将上清液转移到Beckman聚碳酸酯管(在70Ti型转子中填充水平为16.5ml)。 使用Type 70 Ti转子,以44,000 x g(24,453 rpm)高速旋转30分钟,以沉淀微粒体。
    7. 将微粒体重悬于0.5ml裂解缓冲液中,然后用补充有蛋白酶抑制剂的2ml洋地黄皂苷的14ml免疫沉淀缓冲液稀释。
    8. 膜蛋白可以通过在4℃下裂解裂解物1.5小时来溶解。 通过在44,000×g离心30分钟(在74Ti型转子中为24,453rpm)除去未溶解的材料。

  2. FLAG融合蛋白免疫沉淀
    1. 每个反应使用300微升的凝胶悬浮液(〜150微升的填充凝胶体积)。
    2. 将ANTI-FLAG M2亲和凝胶完全悬浮在小瓶中,并将其转移到15ml锥形管中。
    3. 在400×g离心树脂1分钟。 在处理样品前等待1-2分钟。 用凝胶装载移液管吸头除去上清液。
    4. 用5ml含0.1%毛地黄皂苷的免疫沉淀缓冲液洗涤填充的凝胶4x。
    5. 将约15ml细胞裂解物加入到ANTI-FLAG树脂中。 将沉淀在4℃下旋转3小时或过夜。
    6. 用10ml含0.1%毛地黄皂苷的免疫沉淀缓冲液洗涤树脂4x。 将珠子转移到Eppendorf管中
  3. 洗脱FLAG融合蛋白
    用3x FLAG肽洗脱。 使用这种方法,洗脱效率非常高。
    1. 制备3x FLAG洗脱缓冲液。 加入150μl1×免疫沉淀缓冲液与0.25%毛地黄皂苷和1μg/μl3x FLAG肽。
    2. 加入150μl的3x FLAG洗脱缓冲液到试管中的树脂。
    3. 孵育样品轻轻摇动30分钟,在4℃。
    4. 在400〜1000×g离心树脂1分钟。 将上清液转移到新鲜试管中。
    5. 用另一150μl含有0.25%洋地黄皂苷和0.1μg/μl3×FLAG肽的3×FLAG洗脱缓冲液重复洗脱。 合并洗脱液。

食谱

  1. 裂解缓冲液
    裂解缓冲液
    1 L
    2x  缓冲液,无洗涤剂
    50 mM HEPES(FW 238.3)

    23.83克
    50mM KOAc * (FW 98.14)

    9.814克
    2mM Mg(OAc)2

    4ml的1M Mg(OAc)2
    1mM CaCl 2

    20ml的0.1M CaCl 2·h/v
    200mM山梨醇(FW 182.17)

    72.87克
    1mM NaF (Ser/Thr磷酸酶抑制剂)

    0.084克
    0.3mM Na 3+ VO 4(Tyr磷酸酶抑制剂)

    0.1103 g

  2. 免疫沉淀缓冲液
    免疫沉淀缓冲液
    1 L
    2x无缓冲液的缓冲液
    50mM HEPES-KOH(pH 6.8)

    23.83克
    50mM KOAc *(FW 98.14)

    9.814克
    2mM Mg(OAc)2

    4ml的1M Mg(OAc)2
    1mM CaCl 2

    20ml的0.1M CaCl 2·h/v
    15%甘油(v/v)
    300ml 100%甘油
    加入Mili-Q H 2 O至最终体积,使用0.5M KOH调节至pH = 6.8。
    在使用前,每50ml免疫沉淀缓冲液中加入以下蛋白酶抑制剂:1片完全片剂,无EDTA的蛋白酶抑制剂(在称重纸上压碎片剂)。
    加入100μl的0.5M PMSF的DMSO溶液。
  3. 50ml裂解缓冲液 25ml 2x裂解缓冲液 25ml H 2 O 2 / 1 EDTA游离蛋白酶抑制剂片剂
    100μl0.5M PMSF的DMSO溶液
  4. 14ml含有2%洋地黄毒苷的IP缓冲液
    7ml 2×IP缓冲液
    2.8ml 10%的洋地黄皂苷
    1ml EDTA游离蛋白酶抑制剂片剂
    28μl0.5M PMSF的DMSO溶液
    4.2ml H 2 O x /
  5. 20ml含有0.1%洋地黄毒苷的IP缓冲液
    10ml 2×IP缓冲液
    0.2ml的10%毛地黄皂苷
    9.8ml H 2 O 2 / 蛋白酶抑制剂(20μlPMSF,亮抑酶肽和胃蛋白酶抑制剂)
  6. 40ml含有0.1%毛地黄皂苷的IP缓冲液
    20ml 2×IP缓冲液
    0.4ml 10%的洋地黄皂苷
    19.6ml H 2 O 2 / 蛋白酶抑制剂(40μlPMSF,亮抑酶肽和胃蛋白酶抑制剂)
  7. 150μl含有1μg/μl3×FLAG肽的洗脱缓冲液
    75μl2x IP缓冲液
    3.75μl的10%毛地黄皂苷
    30μl5mg/ml 3×FLAG肽
    41.25μl的H 2 O 2 /
  8. 制备150μl含有0.1μg/μl3×FLAG肽的洗脱缓冲液
    75μl2x IP缓冲液
    3.75μl的10%毛地黄皂苷
    3μl5mg/ml 3×FLAG肽
    68.25μl的H 2 O x/b

致谢

该协议已经在约翰霍普金斯医学院的Espenshade实验室中修改和改编。 资助支持不同的项目,使用这个协议来自NIH - 国家心脏,肺和血液研究所,国家过敏和传染病研究所,胰腺癌行动网络和美国心脏协会。

参考文献

  1. Breslow,D.K.,Collins,S.R.,Bodenmiller,B.,Aebersold,R.,Simons,K.,Shevchenko,A.,Ejsing,C.S。和Weissman,J.S。(2010)。 Orm家族蛋白介导鞘脂内稳态。 自然 463 7284):1048-1053。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tong, Z. (2011). Large Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast. Bio-protocol Bio101: e12. DOI: 10.21769/BioProtoc.12;
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