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Abstract

Biochemistry

Proteína de captura por afinidade complexos a partir de células de mamíferos Cryomilled

Published: December 9th, 2016

DOI:

10.3791/54518

1Laboratory of Cellular and Structural Biology, The Rockefeller University, 2Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine

ERRATUM NOTICE

Important: There has been an erratum issued for this article. Read more …

captura de afinidade é uma técnica eficaz para isolar complexos de proteínas endógenas para um estudo mais aprofundado. Quando usado em conjunto com um anticorpo, esta técnica é também frequentemente referida como imunoprecipitação. de captura por afinidade pode ser aplicado em uma escala de bancada e num contexto de alto rendimento. Quando acoplada a espectrometria de massa de proteína, captura de afinidade provou ser um burro de carga da análise interactome. Embora existam muitas maneiras potencialmente para executar os vários passos envolvidos, os seguintes protocolos de implementar os nossos métodos favorecidas. Duas características são distintos: o uso de pó de células cryomilled para produzir extractos celulares e esferas paramagnéticas acopladas a anticorpo como meio de afinidade. Em muitos casos, obtivemos resultados superiores aos obtidos com mais práticas convencionais de captura de afinidade. Cryomilling evita numerosos problemas associados com outras formas de rebentamento celular. Ele fornece quebra eficiente do material, evitando ao mesmo tempo denatURAÇÃO problemas associados com o aquecimento ou a formação de espuma. Ele retém a proteína nativa concentração até ao ponto de extracção, mitigando dissociação macromolecular. Reduz o tempo de proteínas extraídas passar em solução, limitando actividades enzimáticas deletérios, e pode reduzir a adsorção não específica de proteínas por meio de afinidade. Micron escala meios de afinidade magnética têm se tornado mais comum nos últimos anos, cada vez mais substituindo o agarose- tradicional e mídia baseada em Sepharose. principais benefícios de meios magnéticos incluem adsorção de proteínas não-específicas tipicamente inferior; nenhum limite de exclusão de tamanho, porque proteína de ligação ocorre na superfície do grânulo em vez de dentro dos poros; e facilidade de manipulação e manuseio usando ímãs.

Erratum

Erratum: Protein Complex Affinity Capture from Cryomilled Mammalian Cells

A correction was made to: Protein Complex Affinity Capture from Cryomilled Mammalian Cells. The References section has been updated from:

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  2. Rosenberg, I. M. Electrophoretic Techniques. Protein Analysis and Purification. (4), 63–117 (2005).
  3. Ornstein, L. Disc Electrophoresis. I. Background and Theory. Ann N Y Acad Sci. 121 (A2), 321–349 (1964).
  4. Davis, B. J. Disc Electrophoresis. II. Method and Application to Human Serum Proteins. Ann N Y Acad Sci. 121, 404–427 (1964).
  5. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227 (5259), 680–685 (1970).
  6. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature protocols. 1 (6), 2856–2860 (2006).
  7. DeGrasse, J. A., Kalkum, M., Krutchinsky, A.N., Padovan, J. C., & Zhang, W. MALDI Sample Preparation. rockefeller.edu at <http://prowl.rockefeller.edu/protocols/in-gel-digestion.html> (2006).
  8. Lubas, M., Christensen, M. S., et al. Interaction profiling identifies the human nuclear exosome targeting complex. Mol Cell. 43 (4), 624–637 (2011).
  9. Tackett, A. J., DeGrasse, J. A., Sekedat, M. D., Oeffinger, M., Rout, M. P., & Chait, B. T. I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions. J Proteome Res. 4 (5), 1752–1756 (2005).
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  11. Trinkle-Mulcahy, L., Boulon, S., et al. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol. 183 (2), 223–239 (2008).
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  13. Armean, I. M., Lilley, K. S., & Trotter, M. W. B. Popular computational methods to assess multiprotein complexes derived from label-free affinity purification and mass spectrometry (AP-MS) experiments. Mol Cell Proteomics. 12 (1), 1–13 (2013).
  14. Mellacheruvu, D., Wright, Z., et al. The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods. (2013).
  15. Cheeseman, I. M., & Desai, A. A combined approach for the localization and tandem affinity purification of protein complexes from metazoans. Sci. STKE 2005 (266), pl1 (2005).
  16. Goldberg, S. Mechanical/physical methods of cell disruption and tissue homogenization. Methods in Molecular Biology. 424 (Chapter 1), 3–22 (2008).
  17. Grabski, A. C. Advances in preparation of biological extracts for protein purification. Methods in Enzymology. 463 (C), 285–303 (2009).
  18. Dhabaria, A., Cifani, P., Reed, C., Steen, H., & Kentsis, A. A High-Efficiency Cellular Extraction System for Biological Proteomics. J Proteome Res. 14 (8), 3403–3408 (2015).
  19. Glatter, T., Ahrné, E., & Schmidt, A. Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells. J Proteome Res. 15 (2), 679 (2016).
  20. Zhao, Q.-Q., Yamada, S., & Jimbo, G. The Mechanism and Grinding Limit of Planetary Ball Milling. KONA. 7, 29–36 (1989).
  21. Sheng-Yong, L., Qiong-Jing, M., Zheng, P., Xiao-Dong, L., & Jian-Hua, Y. Simulation of ball motion and energy transfer in a planetary ball mill. Chinese Phys. B 21 (7), 078201 (2012).
  22. Fulton, A. B. How crowded is the cytoplasm? Cell. 30 (2), 345–347 (1982).
  23. Ellis, R. J. Macromolecular crowding: obvious but underappreciated. Trends Biochem Sci. 26 (10), 597–604 (2001).
  24. Kalkum, M. Using the Retsch MM301 Ball Mill for Cryogenic Disruption of Yeast Cells. 2 City of Hope, (2003).
  25. Staley, J. Making Whole Cell Extract of Saccharomyces cerevisiae cells using the Retsch MM301 Ball Mill. Retsch GmbH: (2005).
  26. Bell, A. W., Nilsson, T., Kearney, R. E., & Bergeron, J. J. M. The protein microscope: Incorporating mass spectrometry into cell biology. Nat Methods. 4 (10), 783–784 (2007).
  27. Zhao, X., Li, G., & Liang, S. Several affinity tags commonly used in chromatographic purification. J Anal Methods Chem. 2013 (1), 581093–8 (2013).
  28. Waugh, D. S. An overview of enzymatic reagents for the removal of affinity tags. 80 (2), 283–293 (2011).
  29. Williams, R. J., & Lyman, C. M. A neutral buffered standard for hydrogen ion work and accurate titrations which can be prepared in one minute. J Am Chem Soc. 54 (5), 1911–1912 (1932).
  30. Johnson, M. Detergents: Triton X-100, Tween-20, and More. labome.com. 3 (163) (2013).
  31. Deppert, W. R., & Lukaĉin, R. Buffers and Additives. Journal of Chromatography Library. 61 (C), 839–862 (1999).
  32. Ugwu, S. O., & Apte, S. P. The Effect of Buffers on Protein Conformational Stability. Pharmaceutical Technology. 28 (3), 86–108 (2004).
  33. Linke, D. Detergents: an overview. Methods in Enzymology. 463 (34), 603–617 (2009).
  34. Zhang, J. Protein-Protein Interactions in Salt Solutions. Protein-Protein Interactions Computational and Experimental Tools. (18), 359–376 (2012).

to:

  1. Ball Mills - Guidelines for sample amount and ball charge. 1–3at <http://www.retsch.com/products/milling/ball-mills/planetary-ball-mill-pm-100/information-downloads/> Retsch GmbH (2014).
  2. Cristea, I. M., & Chait, B. T. Conjugation of magnetic beads for immunopurification of protein complexes. Cold Spring Harbor Protocols. 2011 (5) (2011).
  3. Rosenberg, I. M. Electrophoretic Techniques. Protein Analysis and Purification. (4), 63–117 (2005).
  4. Ornstein, L. Disc Electrophoresis. I. Background and Theory. Ann N Y Acad Sci. 121 (A2), 321–349 (1964).
  5. Davis, B. J. Disc Electrophoresis. II. Method and Application to Human Serum Proteins. Ann N Y Acad Sci. 121, 404–427 (1964).
  6. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227 (5259), 680–685 (1970).
  7. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature protocols. 1 (6), 2856–2860 (2006).
  8. DeGrasse, J. A., Kalkum, M., Krutchinsky, A.N., Padovan, J. C., & Zhang, W. MALDI Sample Preparation. rockefeller.edu at <http://prowl.rockefeller.edu/protocols/in-gel-digestion.html> (2006).
  9. Lubas, M., Christensen, M. S., et al. Interaction profiling identifies the human nuclear exosome targeting complex. Mol Cell. 43 (4), 624–637 (2011).
  10. Tackett, A. J., DeGrasse, J. A., Sekedat, M. D., Oeffinger, M., Rout, M. P., & Chait, B. T. I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions. J Proteome Res. 4 (5), 1752–1756 (2005).
  11. Wang, X., & Huang, L. Identifying dynamic interactors of protein complexes by quantitative mass spectrometry. Mol Cell Proteomics. 7 (1), 46–57 (2008).
  12. Trinkle-Mulcahy, L., Boulon, S., et al. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol. 183 (2), 223–239 (2008).
  13. Boulon, S., Ahmad, Y., et al. Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol Cell Proteomics. 9 (5), 861–879 (2010).
  14. Armean, I. M., Lilley, K. S., & Trotter, M. W. B. Popular computational methods to assess multiprotein complexes derived from label-free affinity purification and mass spectrometry (AP-MS) experiments. Mol Cell Proteomics. 12 (1), 1–13 (2013).
  15. Mellacheruvu, D., Wright, Z., et al. The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods. (2013).
  16. Cheeseman, I. M., & Desai, A. A combined approach for the localization and tandem affinity purification of protein complexes from metazoans. Sci. STKE 2005 (266), pl1 (2005).
  17. Deppert, W. R., & Lukaĉin, R. Buffers and Additives. Journal of Chromatography Library. 61 (C), 839–862 (1999).
  18. Ugwu, S. O., & Apte, S. P. The Effect of Buffers on Protein Conformational Stability. Pharmaceutical Technology. 28 (3), 86–108 (2004).
  19. Linke, D. Detergents: an overview. Methods in Enzymology. 463 (34), 603–617 (2009).
  20. Zhang, J. Protein-Protein Interactions in Salt Solutions. Protein-Protein Interactions Computational and Experimental Tools. (18), 359–376 (2012).
  21. Goldberg, S. Mechanical/physical methods of cell disruption and tissue homogenization. Methods in Molecular Biology. 424 (Chapter 1), 3–22 (2008).
  22. Grabski, A. C. Advances in preparation of biological extracts for protein purification. Methods in Enzymology. 463 (C), 285–303 (2009).
  23. Dhabaria, A., Cifani, P., Reed, C., Steen, H., & Kentsis, A. A High-Efficiency Cellular Extraction System for Biological Proteomics. J Proteome Res. 14 (8), 3403–3408 (2015).
  24. Glatter, T., Ahrné, E., & Schmidt, A. Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells. J Proteome Res. 15 (2), 679 (2016).
  25. Zhao, Q.-Q., Yamada, S., & Jimbo, G. The Mechanism and Grinding Limit of Planetary Ball Milling. KONA. 7, 29–36 (1989).
  26. Sheng-Yong, L., Qiong-Jing, M., Zheng, P., Xiao-Dong, L., & Jian-Hua, Y. Simulation of ball motion and energy transfer in a planetary ball mill. Chinese Phys. B 21 (7), 078201 (2012).
  27. Fulton, A. B. How crowded is the cytoplasm? Cell. 30 (2), 345–347 (1982).
  28. Ellis, R. J. Macromolecular crowding: obvious but underappreciated. Trends Biochem Sci. 26 (10), 597–604 (2001).
  29. Kalkum, M. Using the Retsch MM301 Ball Mill for Cryogenic Disruption of Yeast Cells. 2 City of Hope, (2003).
  30. Staley, J. Making Whole Cell Extract of Saccharomyces cerevisiae cells using the Retsch MM301 Ball Mill. Retsch GmbH: (2005).
  31. Bell, A. W., Nilsson, T., Kearney, R. E., & Bergeron, J. J. M. The protein microscope: Incorporating mass spectrometry into cell biology. Nat Methods. 4 (10), 783–784 (2007).
  32. Zhao, X., Li, G., & Liang, S. Several affinity tags commonly used in chromatographic purification. J Anal Methods Chem. 2013 (1), 581093–8 (2013).
  33. Waugh, D. S. An overview of enzymatic reagents for the removal of affinity tags. 80 (2), 283–293 (2011).

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