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Tandem affinity purification (TAP) (Pugi et al.,2001; Rigaut et al., 1999) is a method that uses a tagging approach of a target protein of interest for a two-step purification scheme in order to pull down protein complexes under native conditions and expression levels. The TAP tag consists of three components: a calmodulin-binding peptide, a Tobacco etch virus (TEV) protease cleavage site and Protein A which is an immunoglobulin G (IgG)-binding domain. This protocol was modified from the original methodology used in yeast cells(Pugi et al.,2001; Rigaut et al., 1999) for isolation of protein complexes from Drosophila heads and ovaries expressing a TAP tagged protein of interest. To determine in vivo binding partners of the Drosophila fragile X protein (dFMR1), we developed a transgenic strain of flies expressing a recombinant form of dFMR1 with a carboxy-terminal TAP tag (Tsai and Carstens, 2006). To ensure that the construct was expressed at wild-type levels, we engineered this form of the tagged protein in the context of a genomic rescue construct that rescued a mutant sterility phenotype. The purification process was performed using mild conditions to maintain native protein interactions. For TAP methods in Drosophila S2 cell culture, we have successfully used a protocol previously published by Tsai and Carstens (Tsai and Carstens, 2006; Bhogal et al., 2011).

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Tandem Affinity Purification in Drosophila Heads and Ovaries
在果蝇头部和卵巢中进行串联亲和纯化

发育生物学 > 细胞信号传导 > TAP标记
作者: Anita Pepper
Anita PepperAffiliation: Department of Genetics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a75
Balpreet Bhogal
Balpreet BhogalAffiliation: Department of Genetics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a76
 and Thomas Jongens
Thomas JongensAffiliation: Department of Genetics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA
For correspondence: jongens@mail.med.upenn.edu
Bio-protocol author page: a77
Vol 2, Iss 15, 8/5/2012, 6840 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.245

[Abstract] Tandem affinity purification (TAP) (Pugi et al.,2001; Rigaut et al., 1999) is a method that uses a tagging approach of a target protein of interest for a two-step purification scheme in order to pull down protein complexes under native conditions and expression levels. The TAP tag consists of three components: a calmodulin-binding peptide, a Tobacco etch virus (TEV) protease cleavage site and Protein A which is an immunoglobulin G (IgG)-binding domain. This protocol was modified from the original methodology used in yeast cells(Pugi et al.,2001; Rigaut et al., 1999) for isolation of protein complexes from Drosophila heads and ovaries expressing a TAP tagged protein of interest. To determine in vivo binding partners of the Drosophila fragile X protein (dFMR1), we developed a transgenic strain of flies expressing a recombinant form of dFMR1 with a carboxy-terminal TAP tag (Tsai and Carstens, 2006). To ensure that the construct was expressed at wild-type levels, we engineered this form of the tagged protein in the context of a genomic rescue construct that rescued a mutant sterility phenotype. The purification process was performed using mild conditions to maintain native protein interactions. For TAP methods in Drosophila S2 cell culture, we have successfully used a protocol previously published by Tsai and Carstens (Tsai and Carstens, 2006; Bhogal et al., 2011).

[Abstract] 该实验方案的中文版正在准备中...

Materials and Reagents

  1. 1x Phosphate buffered saline (PBS)
  2. Fly sieves: 106, 355, 600 and 850 mm
  3. IgG Sepharose Fast Flow Beads (GE Healthcare Dharmacon, catalog number:  17-0969-01 )
  4. AcTEV Protease (Life Technologies, Invitrogen™, catalog number:  12575-015 )
  5. Calmodulin Sepharose 4B (GE Healthcare Dharmacon, catalog number: 17-0529-01 )
  6. Micro Bio-Spin column (Bio-Rad Laboratories, catalog number: 732-6204 )
  7. NuPAGE Novex 4-12% Bis-Tris gel (Life Technologies, catalog number: NP0321 )
  8. Silverquest Silver Staining Kit (Life Technologies, catalog number: LC6070 ), Colloidal Blue Staining Kit (Life Technologies, catalog number: LC6025 ) or Coomassie Blue.
  9. Beta-mercaptoethanol
  10. 1 M CaCl2
  11. Hepes
  12. MgCl2
  13. KCl
  14. NP-40
  15. DTT
  16. Na3VO4
  17. EDTA
  18. EGTA
  19. NaF
  20. Glycerol
  21. Mg-acetate
  22. Imidazole
  23. Bouwmeester's buffer (see Recipes)
  24. Buffer A (see Recipes)
  25. Buffer B (see Recipes)
  26. Buffer C (see Recipes)
  27. TEV Cleavage buffer (see Recipes)
  28. Calmodulin binding buffer (see Recipes)
  29. Calmodulin blution buffer  (see Recipes)

Equipment

  1. Benchtop centrifuge (that fits 1.5 ml microcentrifuge tubes )
  2. 1.5 ml microcentrifuge tubes
  3. Nutator
  4. Vortexer
  5. Ceramic mortar and pestle
  6. Dounce homogenizer
  7. Disposable pestles for 1.5 ml tubes
  8. Beckman table top centrifuge (that fits 15 ml and 50 ml conical tubes and can be cooled to 4 °C)
  9. Sorvall with a Beckman JA-20 rotor that is capable to cool to 4 °C
  10. Mass Spectrometry Facility

Procedure

  1. Isolation of protein complexes
    1. For protein complex isolation from drosophila ovaries:
      1. Feed (1-3 day old) flies expressing your TAP-tagged protein for three days with yeast paste.
      2. Dissect ovaries in 1x PBS, collect in a 1.5 ml microcentrifuge tube, and freeze at -80 °C. Collect more than 200 pairs of ovaries. Ovaries can be collected following several independent rounds of dissection and frozen to amass an appropriate amount.
      3. Homogenize ovaries in 1 ml Bouwmeester's buffer using disposable pestles that fit 1.5 ml microcentrifuge tube.
      4. Incubate on ice for 20 min and transfer to Beckman centrifuge tubes (1/2 x 2 inch).
      5. Spin tubes at 4 °C, 20,000 x g for 15 min.
      6. Remove and save supernatant on ice. This is your cytoplasmic extract.
      7. Spin tubes at 4 °C, 100,000 x g for 45 min to remove remaining extract following additional spin.
      8. Remove and save supernatant on ice. This is your nuclear extract (can combine the extracts from step A6 and A8, if you want total extracts, or keep separate).
      9. Take out 20 μl from each of your isolated extracts as a lysate control (control A). Freeze in liquid nitrogen and store at -80 °C. Go to step #a in Section 2: Pulldown of protein complexes.
    2. For protein complex isolation from drosophila heads:
      1. Freeze flies expressing TAP-tagged protein of interest at –80 °C. You will need at least 50 ml of frozen flies to begin. Weigh starting materials on a standard scale and be sure to have at least 1 gram of heads.
      2. Chill sieves in liquid nitrogen in preparation for head popping procedure.
      3. Pop heads off using liquid nitrogen and the proper sized meshes (http://cmgm.stanford.edu/~vesicle/protocols/droshead.html). Keep purified head samples on dry ice or at -80 °C until you are ready for the next step.
      4. Pre-freeze mortar and pestle using liquid nitrogen. Add heads and liquid nitrogen to chilled mortar and pestle and grind sample on dry ice, continuing to add liquid nitrogen until you have a fine powder.
      5. When you have fine powder, add more liquid nitrogen to pour out powder into a 50 ml falcon tube.
      6. Let liquid nitrogen de-gas in uncovered tube for 5-10 min.
      7. Add 2 ml bouwmeester buffer to powder, transfer to a dounce homogenizer and dounce homogenize 10 times. Transfer to several 1.5 ml microcentrifuge tubes. Spin 15 min, 15,000 x g at 4 °C, remove supernatant and repeat spin to remove as much as supernatant as possible.
      8. Take out 20 μl as a lysate control from each sample (control A). Snap freeze in liquid nitrogen and store at -80 °C.

  2. Pulldown of protein complexes
    1. Prepare 200 μl IgG Sepharose Fast Flow beads in a 15 ml falcon tube and wash twice with 500 μl buffer B (add 100 μl PIC and 5 μl DTT fresh to 10 ml buffer B). After each wash with buffer B, spin at 4 °C, 100 x g for 2 min in a Beckman table top centrifuge and remove supernatant.
    2. Add lysate and rotate overnight at 4 °C on a nutator.
    3. After overnight incubation, spin at 4 °C, 100 x g for 5 min in a Beckman table top centrifuge and remove supernatant. Save 20 μl of supernatant for post-incubation control (control B).
    4. Wash beads 3 times in 1 ml Buffer B. Spin 4 °C, 100 x g for 2 min after each wash in a Beckman table top centrifuge.
    5. Wash beads 4 times in 1 ml Buffer C. Spin 4°C, 100 x g for 2 min after each wash in a Beckman table top centrifuge.
    6. Wash twice in 1 ml TEV cleavage buffer. Spin 4 °C, 100 x g for 2 min after each wash in a Beckman table top centrifuge. Remove 10 μl beads as a control (control C).
    7. Add 400 μl TEV cleavage buffer and 4 μl AcTEV protease to beads and transfer to a 1.5 ml microcentrifuge tube. Rotate overnight at 4 °C on a nutator.
    8. Spin 300 x g, 1 min at 4 °C in a bench top centrifuge.
    9. Take off supernatant and add 0.6 μl 1 M CaCl2 per 200 μl supernatant (usually 1.2 μl for 400 μl total supernatant).
    10. Prepare a 1.5 ml microcentrifuge tube with 100 μl Calmodulin sepharose resin and wash twice with 500 μl Calmodulin binding buffer. Spin 2 min, 1,500 rpm, 4 °C after each wash.
    11. Add 3x volume Calmodulin binding buffer to supernatant and add this to prepared calmodulin resin. Rotate 3-4 h at 4 °C.
    12. Spin 2 min, 1,500 rpm, 4 °C.
    13. Wash three times with 500 μl Calmodulin binding buffer. Spin 2 min, 1,500 rpm, 4 °C after each wash.
    14. Elute TAP-tagged protein with Calmodulin elution buffer using preferred strategy.
    15. Run eluate on a 4-12% Bis-Tris 10 well gel.
    16. Run controls and protein ladder on a separate gel from final eluate so there is no contamination.
    17. Stain with either Coomassie Blue, Colloidal Blue Staining Kit, or Silverquest Kit. Cut out bands of interest and send to the Mass Spectrometry Facility.

Recipes

Note: Make buffers without Protease Inhibitor Cocktail without EDTA (PIC w/o EDTA), Na3VO4, CaCl2 and/or DTT. These should be added fresh right before use.

  1. Buffer A (20 ml)
    See note above
    Stock and concentration volume Final concentration
    1 M Hepes (pH 7.9) 200 ml 10 mM Hepes
    1 M MgCl2 30 ml 1.5 mM MgCl2
    2 M KCl 100 ml 10 mM KCl
    1 M DTT 10 ml 0.5 mM DTT
    PIC w/o EDTA 20 ml 1x
    H2O to 20 ml  
  2. Bouwmeester's buffer (20 ml)
    Filter Sterilize and store at 4 °C.
    See note above

    Stock and concentration volume Final concentration
    1 M Tris-HCl (pH 7.5) 1 ml 50 mM Tris-HCl
    5 M NaCl 2.5 ml 125 mM NaCl
    100% glycerol 1 ml 5% glycerol
    100% NP-40 80 ml 0.2% NP-40
    1 M MgCl2 30 ml 1.5 mM MgCl2
    1 M DTT 20 ml 1 mM DTT
    1 M NaF 500 ml 25 mM NaF
    200 mM Na3VO4 100 ml 1 mM Na3VO4
    500 mM EDTA 40 ml 1 mM EDTA
    2 mM EGTA 80 ml 500 mM EGTA
    PIC w/o EDTA 20 ml 1x
    H2O to 20 ml  
  3. Buffer B (20 ml)
    Make fresh on the day of experiment
    See note above
    Stock and concentration volume Final concentration
    1 M Hepes (pH 7.9) 400 ml 20 mM Hepes
    87% glycerol 4.6 ml 20% glycerol
    100% NP-40 200 ml 0.5% NP-40
    2 M KCl 2.5 ml 200 mM KCl
    1 M DTT 10 ml 0.5 mM DTT
    500 mM EDTA 40 ml 1 mM EDTA
    20 mM EGTA 80 ml 500 mM EGTA
    PIC w/o EDTA 200 ml 1x
    H2O to 20 ml  
  4. Buffer C (20 ml)
    See note above
    Stock and concentration volume Final concentration
    1 M Hepes (pH 7.9) 400 ml 20 mM Hepes
    87% glycerol 4.6 ml 20% glycerol
    100% NP-40 200 ml 0.5% NP-40
    2 M KCl 2.5 ml 200 mM KCl
    1 M DTT 10 ml 0.5 mM DTT
    H2O to 20 ml  
  5. TEV cleavage buffer (100 ml)
    Filter sterilize
    See note above
    Stock and
    concentration
    volume Final concentration
    1 M Tris-HCl (pH 8.0) 1 ml 10 mM Tris-HCl
    5 M NaCl 3 ml 150 mM NaCl
    20% NP-40 500 ml 0.1% NP-40
    1 M DTT 50 ml 0.5 mM EDTA
    H2O to 100 ml  
  6. Calmodulin binding buffer (100 ml)
    Stock and
    concentration
    Volume Final concentration
    1 M Tris-HCl (pH 8.0) 1 ml 10 mM Tris-HCl
    Beta-mercaptoethanol 69.7 ml 10 mM
    Beta-mercaptoethanol
    5 M NaCl 3 ml 150 mM NaCl
    1 M Mg-acetate 100 ml 1 mM Mg-acetate
    1 M Imidazole 100 ml 1 mM Imidazole
    1 M CaCl2 200 ml 2 mM CaCl2
    20% NP-40 500 ml 0.1% NP-40
    H2O to 100 ml  
  7. Calmodulin elution buffer (100 ml)
    Stock and
    concentration
    Volume Final concentration
    1 M Tris-HCl (pH 8.0) 1 ml 10 mM Tris-HCl
    Beta-mercaptoethanol 69.7 ml 10 mM Beta-mercaptoethanol
    5 M NaCl 3 ml 150 mM NaCl
    1 M Mg-acetate 100 ml 1 mM Mg-acetate
    1 M Imidazole 100 ml 1 mM Imidazole
    20% NP-40 500 ml 0.1% NP-40
    2 mm EGTA 400 ml 500 mM EGTA
    H2O to 100 ml  

Acknowledgments

This protocol was modified from the original methodology used in yeast cells (Pugi et al., 2001; Rigaut et al., 1999).

References

  1. Bhogal, B., Jepson, J. E., Savva, Y. A., Pepper, A. S., Reenan, R. A. and Jongens, T. A. (2011). Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein. Nat Neurosci 14(12): 1517-1524.
  2. Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M. and Seraphin, B. (2001). The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24(3): 218-229.
  3. Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M. and Seraphin, B. (1999). A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17(10): 1030-1032.
  4. Tsai, A. and Carstens, R. P. (2006). An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach. Nat Protoc 1(6): 2820-2827.

材料和试剂

  1. 1x磷酸盐缓冲盐水(PBS)
  2. 飞筛:106,355,600和850 mm
  3. IgG Sepharose Fast Flow Beads(GE Healthcare Dharmacon,目录号:17-0969-01)
  4. AcTEV蛋白酶(Life Technologies,Invitrogen TM,目录号:12575-015)
  5. 钙调蛋白琼脂糖4B(GE Healthcare Dharmacon,目录号:17-0529-01)
  6. Micro Bio-Spin柱(Bio-Rad Laboratories,目录号:732-6204)
  7. NuPAGE Novex 4-12%Bis-Tris凝胶(Life Technologies,目录号:NP0321)
  8. Silverquest银染色试剂盒(Life Technologies,目录号:LC6070),胶体蓝染色试剂盒(Life Technologies,目录号:LC6025)或考马斯蓝。
  9. β-巯基乙醇
  10. 1 M CaCl 2
  11. Hepes
  12. MgCl 2
  13. KCl
  14. NP-40
  15. DTT
  16. Na 3 3 VO 4
  17. EDTA
  18. EGTA
  19. NaF
  20. 甘油
  21. 醋酸镁
  22. 咪唑
  23. Bouwmeester的缓冲区(见Recipes)
  24. 缓冲液A(参见配方)
  25. 缓冲液B(参见配方)
  26. 缓冲液C(参见配方)
  27. TEV裂解缓冲液(见配方)
  28. 钙调蛋白结合缓冲液(参见配方)
  29. 钙调素清除缓冲区(参见配方)

设备

  1. 台式离心机(适用于1.5 ml微量离心管)
  2. 1.5ml微量离心管
  3. 营养器
  4. Vortexer
  5. 陶瓷砂浆和杵
  6. Dounce匀浆器
  7. 1.5ml管的一次性杵
  8. Beckman台式离心机(适用于15 ml和50 ml锥形管,可以冷却至4°C)
  9. Sorvall与Beckman JA-20转子,能够冷却至4°C
  10. 质谱设备

程序

  1. 蛋白复合物的分离
    1. 对于从果蝇卵巢中分离蛋白质复合物:
      1. 饲料(1-3天龄)用酵母糊状物表达TAP-标记的蛋白质三天。
      2. 解剖卵巢在1×PBS,收集在1.5毫升微量离心管,并在-80℃冷冻。 收集超过200对卵巢。 可以在几轮独立的解剖后收集卵巢,并冷冻以收集适当的量。
      3. 使用适合1.5ml微量离心管的一次性杵在1ml Bouwmeester's缓冲液中匀浆卵巢。
      4. 在冰上孵育20分钟,并转移到Beckman离心管(1/2 x 2英寸)
      5. 在4℃,20,000×g下旋转管15分钟。
      6. 取出并保存在冰上的上清液。这是你的细胞质提取物。
      7. 在4℃下旋转管,100,000xxg,在另外旋转后45分钟以除去剩余的提取物。
      8. 取出并保存在冰上的上清液。这是你的核提取物(可以组合步骤A6和A8的提取,如果你想要提取物,或保持分离)。
      9. 从每个离体提取物中取出20μl作为裂解物对照(对照A)。在液氮中冷冻并储存在-80℃。转到第2节:蛋白复合物下降的步骤#a
    2. span style ="color:#666666; font-family:Arial,Helvetica,sans-serif; font-size:13.333333015441895px; line-height:20px; text-align:justify; white-space:normal;">复杂的隔离从果蝇头:
      1. 在-80℃下冷冻表达感兴趣的TAP标记的蛋白质。你需要至少50毫升的冷冻苍蝇开始。在标准规模称重起始材料,并确保至少有1克头。
      2. 冷却筛在液氮中准备用于头部爆破程序
      3. 流行音乐使用液氮和适当大小的网格( http://cmgm.stanford。 edu /〜vesicle/protocols/droshead.html )。将纯化的头部样品保存在干冰上或-80°C,直到您准备好下一步。
      4. 使用液氮预冷冻研钵和杵。添加头和液氮到冷冻的研钵和杵,研磨样品在干冰上,继续加入液氮直到你有一个细粉。
      5. 当你有细粉时,加入更多的液氮将粉末倒入50ml的falcon管中
      6. 让液氮在未覆盖的管中脱气5-10分钟
      7. 加入2毫升bouwmeester缓冲液粉末,转移到dounce匀浆器和dounce匀浆10次。 转移到几个1.5毫升微量离心管。 在4℃下旋转15分钟,15,000xg,除去上清液,并重复旋转以除去尽可能多的上清液。
      8. 从每个样品中取出20μl作为裂解物对照(对照A)。 在液氮中快速冷冻并储存在-80℃
  2. 蛋白复合物的下降
    1. 准备200微升IgG Sepharose Fast Flow珠在15毫升falcon管,用500微升缓冲液B(添加100微升PIC和5微升DTT新鲜到10毫升缓冲液B)洗涤两次。用缓冲液B每次洗涤后,在Beckman台式离心机中在4℃,100×g旋转2分钟,除去上清液。
    2. 加入裂解物并在4℃下在营养师上旋转过夜
    3. 孵育过夜后,在4℃下,在Beckman台式离心机中100×g离心5分钟,除去上清液。保存20微升的上清后孵化控制(对照B)。
    4. 在1ml缓冲液B中洗涤珠3次。在Beckman台式离心机中每次洗涤后,在4℃,100×g下旋转2分钟。
    5. 在1ml缓冲液C中洗涤珠4次。在Beckman台式离心机中每次洗涤后,在4℃,100×g下旋转2分钟。
    6. 在1ml TEV裂解缓冲液中洗涤两次。在Beckman台式离心机中每次洗涤后,在4℃,100×g下旋转2分钟。取出10微升珠作为对照(对照C)
    7. 加入400微升TEV裂解缓冲液和4微升AcTEV蛋白酶到珠子,并转移到1.5毫升微量离心管。 在4℃下在旋转器上旋转过夜。
    8. 在台式离心机中在4℃下旋转300分钟,1分钟。
    9. 取出上清液,加入0.6μl1M CaCl 2 /200μl上清液(通常为1.2μl,400μl总上清液)。
    10. 准备1.5毫升微量离心管与100微升钙调蛋白琼脂糖树脂和用500微升钙调蛋白结合缓冲液洗涤两次。 旋转2分钟,1,500rpm,每次洗涤后4℃
    11. 加入3倍体积的钙调蛋白结合缓冲液到上清液,并将其添加到制备的钙调蛋白树脂。 在4℃下旋转3-4小时。
    12. 旋转2分钟,1,500rpm,4℃。
    13. 用500μl钙调蛋白结合缓冲液洗涤三次。 旋转2分钟,1,500rpm,每次洗涤后4℃
    14. 使用优选策略用Calmodulin洗脱缓冲液洗脱TAP标记的蛋白质
    15. 在4-12%Bis-Tris 10孔凝胶上进行洗脱
    16. 运行控制和蛋白梯在独立的凝胶从最终洗脱液,所以没有污染
    17. 用考马斯蓝,胶体蓝染色试剂盒或Silverquest试剂盒染色。 剪切感兴趣的带并发送到质谱设备。

食谱

注意:制备不含蛋白酶抑制剂混合物的缓冲液而不使用EDTA(PIC w/o EDTA),Na 3+,VO 4,CaCl 2和 /或DTT。 这些应在使用前立即添加。

  1. 缓冲液A(20ml)
    见上面的注解
    库存和浓度 最终浓度
    1M Hepes(pH7.9) 200毫升 10mM Hepes
    1 M MgCl 2 30ml 1.5mM MgCl 2
    2 M KCl 100毫升 10mM KCl
    1 M DTT 10毫升 0.5mM DTT
    PIC w/o EDTA 20毫升 1x
    H 2 O 至20mL  
  2. Bouwmeester缓冲液(20ml)
    过滤器消毒并存储在4  °C 。 见上面的注解

    库存和浓度 最终浓度
    1M Tris-HCl(pH7.5) 1ml 50mM Tris-HCl
    5 M NaCl 2.5ml 125mM NaCl
    100%甘油 1ml 5%甘油
    100%NP-40 80ml 0.2%NP-40
    1 M MgCl 2 30ml 1.5mM MgCl 2
    1 M DTT 20毫升 1mM DTT
    1 M NaF 500ml 25mM NaF
    200mM Na 3 VO 4 100毫升 1mM Na 3 VO 4
    500mM EDTA 40ml 1mM EDTA
    2mM EGTA 80ml 500mM EGTA
    PIC w/o EDTA 20毫升 1x
    H 2 O 至20mL  
  3. 缓冲液B(20ml)
    在实验当天更新
    见上面的注解
    库存和浓度 最终浓度
    1M Hepes(pH7.9) 400ml 20mM Hepes
    87%甘油 4.6ml 20%甘油
    100%NP-40 200毫升 0.5%NP-40
    2 M KCl 2.5ml 200mM KCl
    1 M DTT 10毫升 0.5mM DTT
    500mM EDTA 40ml 1mM EDTA
    20mM EGTA 80ml 500mM EGTA
    PIC w/o EDTA 200毫升 1x
    H 2 O 至20mL  
  4. 缓冲液C(20ml)
    见上面的注解
    库存和浓度 最终浓度
    1M Hepes(pH7.9) 400ml 20mM Hepes
    87%甘油 4.6ml 20%甘油
    100%NP-40 200毫升 0.5%NP-40
    2 M KCl 2.5ml 200mM KCl
    1 M DTT 10毫升 0.5mM DTT
    H 2 O 至20mL  
  5. TEV裂解缓冲液(100ml) 过滤灭菌
    见上面的注解
    库存和
    浓度
    最终浓度
    1M Tris-HCl(pH8.0) 1ml 10mM Tris-HCl
    5 M NaCl 3ml 150mM NaCl
    20%NP-40 500ml 0.1%NP-40
    1 M DTT 50毫升 0.5mM EDTA
    H 2 O 至100毫升  
  6. 钙调蛋白结合缓冲液(100ml)
    库存和
    浓度
    最终浓度
    1M Tris-HCl(pH8.0) 1ml 10mM Tris-HCl
    β-巯基乙醇 69.7ml 10 mM
    β-巯基乙醇
    5 M NaCl 3ml 150mM NaCl
    1M Mg-乙酸盐 100毫升 1mM乙酸镁
    1 M咪唑 100毫升 1mM咪唑
    1 M CaCl 2 200毫升 2mM CaCl 2
    20%NP-40 500ml 0.1%NP-40
    H 2 O 至100毫升  
  7. 钙调蛋白洗脱缓冲液(100ml)
    库存和
    浓度
    最终浓度
    1M Tris-HCl(pH8.0) 1ml 10mM Tris-HCl
    β-巯基乙醇 69.7ml 10mMβ-巯基乙醇
    5 M NaCl 3ml 150mM NaCl
    1M Mg-乙酸盐 100毫升 1mM乙酸镁
    1 M咪唑 100毫升 1mM咪唑
    20%NP-40 500ml 0.1%NP-40
    2 mm EGTA 400ml 500mM EGTA
    H 2 O 至100毫升  

致谢

该方案从酵母细胞中使用的原始方法修改而来(Pugi等人,2001; Rigaut等人,1999)。

参考文献

  1. Bhogal,B.,Jepson,J.E.,Savva,Y.A.,Pepper,A.S.,Reenan,R.A。和Jongens,T.A。(2011)。 由果蝇脆弱X智力迟钝蛋白调节dADAR依赖性RNA编辑。 em> Nat Neurosci 14(12):1517-1524。
  2. Puig,O.,Caspary,F.,Rigaut,G.,Rutz,B.,Bouveret,E.,Bragado-Nilsson,E.,Wilm,M.and Seraphin,B。 串联亲和纯化(TAP)方法:蛋白复合物纯化的一般程序。 方法 24(3):218-229。
  3. Rigaut,G.,Shevchenko,A.,Rutz,B.,Wilm,M.,Mann,M。和Seraphin,B。(1999)。 用于蛋白质复合物表征和蛋白质组探索的通用蛋白质纯化方法。 Nat Biotechnol 17(10):1030-1032。
  4. Tsai,A.and Carstens,R.P。(2006)。 使用串联亲和纯化方法在培养的哺乳动物细胞中进行蛋白纯化的优化方案。 Nat Protoc 1(6):2820-2827。
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How to cite this protocol: Pepper, A. S., Bhogal, B. and Jongens, T. (2012). Tandem Affinity Purification in Drosophila Heads and Ovaries. Bio-protocol 2(15): e245. DOI: 10.21769/BioProtoc.245; Full Text



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