欢迎您, 登录 | 注册

首页 | English

X
加载中

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).

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

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, 5905 views, 0 Q&A, How to cite
DOI: http://dx.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).

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.


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



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook