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[Bio101] Small Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast
[Bio101] 小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

In this protocol, we show how to purify membrane proteins from yeast using affinity purification under native conditions at a small scale.

Materials and Reagents

  1. PBS
  2. HEPES
  3. KOAc*
  4. Mg(OAc)2
  5. CaCl2
  6. Sorbitol
  7. Wash buffer
  8. EDTA free protease inhibitors (Roche Diagnostics)
  9. Digitonin (EMD Chemicals)
  10. Protease Inhibitors (DMSO, leupeptin, pepstatin) (Sigma-Aldrich)
  11. ANTI-Flag M2 affinity gel (Sigma-Aldrich)
  12. 3x FLAG peptide (Sigma-Aldrich)
  13. NaF# (Ser/Thr phosphatase inhibitor)
  14. Na3VO4 (Tyr phosphatase inhibitor)
  15. Lysis buffer (see Recipes)
  16. Immunoprecipitation buffer (see Recipes)
  17. Elution buffer (see Recipes)

Equipment

  1. BECKMAN centrifuge and TLA-55 rotor
  2. Acid washed 425-600 glass beads (Sigma-Aldrich)
  3. Beckman centrifuge tubes (Beckman Coulter)

Procedure

  1. Preparation of cell lysate
    1. Collect 25 OD of cells. Wash cells once with 1 ml H2O or PBS at 4 °C. Store at -80 °C if necessary.
    2. Resuspend cells in 150 μl ice-cold immunoprecipitation buffer with 0.1% digitonin and supplemented with protease inhibitors (no DTT) and phosphatase inhibitors. 
    3. Add glass beads below the meniscus. Break cells by vortexing for 10 min in the cold room.
      Note: Digitonin is a less harsh nonionic detergent that permeablizes membranes and can be used to extract membrane proteins in intact forms.
    4. Add 850 μl of immunoprecipitation buffer with 1.16% digitonin plus protease inhibitor (no DTT) to cell lysate, bringing the total volume to 1 ml. This raises the final digitonin concentration to 1%. Membrane proteins are solubilized by nutating lysate at 4 °C for 40 min. Pipette liquid into fresh Beckman centrifuge tubes. 
    5. Centrifuge cell lysate at 100,000 x g for 10 min to clear up unlysed cells, cell nuclei, and membranes. Membranes proteins are extracted from the membranes, hence in the supernatant. Collect supernatant for immunoprecipitation (use TLA-55 rotor, 47,246 rpm). Save 80 μl of the supernatant as input.

  2. FLAG fusion protein immunoprecipitation
    1. Use 50 μl of gel suspension per reaction (~25 μl of packed gel volume). Smaller amounts of resin (~10 μl of packed gel volume, which binds > 1 μg FLAG-tagged protein) can be used.
    2. Thoroughly suspend the ANTI-FLAG M2 affinity gel in the vial. The ratio of suspension to packaged gel volume should be 2:1. 
    3. Immediately transfer 50 μl of the resin in its suspension buffer to a fresh test tube. For resin transfer use a plastic pipette tip with the end cut off.
    4. Centrifuge the resin for 1 min at 400 x g. Wait for 1-2 min before handling the samples. 
    5. Remove the supernatant with a gel-loading pipette tip.
    6. Wash the packed gel 4x with 1 ml of immunoprecipitation buffer with 0.1% digitonin. Be sure that most of the wash buffer is removed and no resin is discarded. This will ensure all of the glycerol is removed before the protein is bound to the gel.
    7. In case of numerous immunoprecipitation samples, wash the resin needed for all samples together. 
    8. After washing, divide the resin according to the number of samples tested. Each wash should be performed with a 1x wash buffer at a volume equal to 20 times the total packed gel volume (e.g., 25 μl packed gel volume, >500 μl wash buffer).
    9. Add 800 μl of cell lysate to the washed resin. If necessary, bring the final volume to 1 ml by adding immunoprecipitation buffer. The volume of cell lysate to be used depends on the expression level of FLAG-tagged protein in the cells. 
    10. For negative control, add only 1 ml of lysis buffer with no protein. 
    11. Agitate samples and control gently on a roller shaker for 2.5 h.
    12. Centrifuge the resin for 1 min at 400 x g. Remove the supernatants with a gel loading pipette tip. Save 80 μl of the supernatant as unbound.
    13. Wash the resin 4x with 1 ml immunoprecipitation buffer.

  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 30 μl of 1x wash buffer with 0.25% digitonin and 2 μg/μl 3x FLAG peptide.
    2. Add 30 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. Be careful not to transfer any resin.

Recipes

  1. Immunoprecipitation buffer
    Immunoprecipitation 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 MgOAc
    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
    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 0.5 M PMSF in DMSO, leupeptin, pepstatin.
  2. To make 1 ml 1x lysis buffer with 0.1% digitonin
    500 μl 2x lysis buffer
    10 μl 10% digitonin
    Protease inhibitors
    490 μl H2O
  3. To make 3.6 ml 1x lysis buffer with 1.16% digitonin
    1.8 ml 2x lysis buffer
    418 μl 10% digitonin
    Protease inhibitors
    1.38 ml H2O
  4. To make 22 ml 1x lysis buffer with 0.1% digitonin
    11 ml 2x lysis buffer
    220 μl 10% digitonin
    Protease inhibitors
    10.7 ml H2O
  5. To make 120 μl elution buffer for 4 samples
    60 μl of 2x lysis buffer
    48 μl of 5 μg/μl 3x FLAG peptide
    3 μl of 10% digitonin
    9 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

在这个协议,我们显示如何纯化膜蛋白从酵母使用亲和纯化在自然条件下小规模。

材料和试剂

  1. PBS
  2. HEPES
  3. KOAc *
  4. Mg(OAc)2
  5. CaCl 2
  6. 山梨醇
  7. 洗涤缓冲液
  8. EDTA游离蛋白酶抑制剂(Roche Diagnostics)
  9. Digitonin(EMD Chemicals)
  10. 蛋白酶抑制剂(DMSO,亮抑酶肽,胃蛋白酶抑制剂)(Sigma-Aldrich)
  11. ANTI-Flag M2亲和凝胶(Sigma-Aldrich)
  12. 3x FLAG肽(Sigma-Aldrich)
  13. NaF (Ser/Thr磷酸酶抑制剂)
  14. (Tyr磷酸酶抑制剂)
  15. 裂解缓冲液(见配方)
  16. 免疫沉淀缓冲液(见配方)
  17. 洗脱缓冲液(见配方)

设备

  1. BECKMAN离心机和TLA-55转子
  2. 酸洗425-600玻璃珠(Sigma-Aldrich)
  3. Beckman离心管(Beckman Coulter)

程序

  1. 细胞裂解液的制备
    1. 收集25 OD细胞。在4℃下用1ml H 2 O或PBS洗涤细胞一次。如有必要,储存于-80°C。
    2. 重悬细胞在150μl冰冷的免疫沉淀缓冲液与0.1%洋地黄皂苷,补充蛋白酶抑制剂(无DTT)和磷酸酶抑制剂。
    3. 在半月板下面添加玻璃珠。通过在冷室中涡旋10分钟破碎细胞 注意:digitonin是一种较不苛刻的非离子洗涤剂,可渗透膜,可用于提取完整形式的膜蛋白。
    4. 添加850微升免疫沉淀缓冲液与1.16%毛地黄皂苷加上蛋白酶抑制剂(无DTT)到细胞裂解液,使总体积为1毫升。这将最终的洋地黄皂苷浓度提高至1%。通过在4℃下使裂解物营养40分钟来溶解膜蛋白。将液体移入新鲜的Beckman离心管中。
    5. 以100,000×g离心细胞裂解物10分钟以清除未裂解的细胞,细胞核和膜。从膜中提取膜蛋白,因此提取上清液。收集上清液用于免疫沉淀(使用TLA-55转子,47,246rpm)。保存80μl上清液作为输入。

  2. FLAG融合蛋白免疫沉淀
    1. 每个反应使用50μl的凝胶悬浮液(〜25μl的填充凝胶体积)。 可以使用更少量的树脂(约10μl的填充凝胶体积,其结合>1μgFLAG标记的蛋白质)。
    2. 将ANTI-FLAG M2亲和凝胶充分悬浮在小瓶中。 悬浮液与包装凝胶体积的比例应为2:1。
    3. 立即将其悬浮液缓冲液中的50μl树脂转移到一个新鲜的试管中。 对于树脂转移,使用塑料移液管末端,末端切除。
    4. 以400×g离心树脂1分钟。 。 在处理样品前等待1-2分钟。
    5. 用凝胶装载移液管吸头除去上清液。
    6. 用1ml含有0.1%毛地黄皂苷的免疫沉淀缓冲液洗涤填充的凝胶4x。确保去除大部分洗涤缓冲液,并且不丢弃树脂。这将确保在蛋白质与凝胶结合之前除去所有甘油。
    7. 在许多免疫沉淀样品的情况下,洗涤所有样品一起所需的树脂。
    8. 洗涤后,根据测试的样品数量分割树脂。每次洗涤应使用体积等于总填充凝胶体积(例如,25μl填充凝胶体积,>500μl洗涤缓冲液)的20倍的1x洗涤缓冲液进行。
    9. 加入800微升细胞裂解液到洗涤的树脂。如有必要,通过加入免疫沉淀缓冲液使终体积达到1ml。使用的细胞裂解物的体积取决于细胞中FLAG标记的蛋白的表达水平。
    10. 对于阴性对照,只加入1ml无蛋白的裂解缓冲液。
    11. 搅拌样品并在滚筒振荡器上温和控制2.5小时。
    12. 在400×g离心树脂1分钟。 用凝胶装载移液管吸头取出上清液。 保存80μl的未结合的上清液。
    13. 用1ml免疫沉淀缓冲液洗涤树脂4x
  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30μl1x洗涤缓冲液与0.25%洋地黄皂苷和2μg/μl3x FLAG肽。
    2. 加入30μl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。 转移上清液到新鲜试管。 小心不要转移任何树脂。

食谱

  1. 免疫沉淀缓冲液
    免疫沉淀缓冲液
    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

    加入4ml 1M MgOAc
    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
    将Mili-Q H 2 O加入到最终体积中,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蛋白酶抑制剂(在称重纸上压碎片剂)。
    加入100微升0.5 M PMSF的DMSO,亮抑酶肽,胃酶抑素。
  2. 用0.1%的洋地黄毒苷制备1ml 1x裂解缓冲液
    500μl2x裂解缓冲液
    10微升10%毛地黄皂苷
    蛋白酶抑制剂
    490μlH 2 O *
  3. 用1.16%的洋地黄皂苷制备3.6ml 1x裂解缓冲液 1.8ml 2×裂解缓冲液 418微升10%毛地黄皂苷
    蛋白酶抑制剂
    1.38ml H 2 O x /
  4. 制备含有0.1%洋地黄毒苷的22ml 1x裂解缓冲液
    11ml 2x裂解缓冲液 220微升10%毛地黄皂苷
    蛋白酶抑制剂
    10.7ml H 2 O 2 /
  5. 对于4个样品制备120μl洗脱缓冲液
    60μl的2x裂解缓冲液
    48μl5μg/μl3×FLAG肽
    3微升10%毛地黄皂苷
    9μlH 2 O 2 /

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). Small Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast. Bio-protocol Bio101: e15. DOI: 10.21769/BioProtoc.15;
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Butnaru Cristian Marian
Institute of Biochemistry
Hello,

i just want to ask a few questions:

1. how did you made the 10% digitonin solution (in water, ethanol...)?
2. for the IP buffer are all the components required (what is the use of sorbitol, NaF#, Na3VO4, CaCl...)?

I tested my digitonin from Santa Cruz and is soluble only in ethanol only at high temperatures, when it cools down or at room temperature it precipitates this is a 1% digitonin buffer
if I would heat up the 2% digitonin lysis buffer in ethanol and quickly add to my cell lysate will it still precipitate ?

every advice is important and precious

thanks!
chris
3/25/2014 1:09:51 AM Reply