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Abstract

Biochemistry

Proteine ​​Complesso affinità Acquisizione da cellule di mammifero 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 …

cattura Affinity è una tecnica efficace per isolare i complessi proteici endogeni per ulteriori studi. Quando utilizzato in combinazione con un anticorpo, questa tecnica viene spesso indicato come immunoprecipitazione. cattura affinità può essere applicato in un banco scala e in un contesto ad alta produttività. Quando accoppiata alla spettrometria di massa proteica, cattura affinità ha dimostrato di essere un cavallo di lavoro di analisi interattoma. Anche se ci sono potenzialmente molti modi per eseguire le numerose fasi, i seguenti protocolli di implementare i nostri metodi favorite. Due caratteristiche sono distintive: l'uso di polvere cellule cryomilled per produrre estratti cellulari e perline paramagnetici anticorpi accoppiati come mezzo affinità. In molti casi, abbiamo ottenuto risultati superiori a quelli ottenuti con altre pratiche cattura affinità convenzionali. Cryomilling evita numerosi problemi associati con altre forme di rottura delle cellule. Esso fornisce efficiente rottura del materiale, evitando denatproblemi confi- connessi con il riscaldamento o la formazione di schiuma. Si mantiene il nativo proteina concentrazione fino al punto di estrazione, mitigare la dissociazione macromolecolare. Si riduce il tempo di proteine ​​estratte passano in soluzione, limitando le attività enzimatiche deleteri, e può ridurre l'adsorbimento non specifico di proteine ​​dal mezzo affinità. Micron scala supporti magnetici affinità sono diventati più comuni negli ultimi anni, sempre più sostituendo la agarose- tradizionale e multimediale Sepharose-based. I vantaggi principali di supporti magnetici includono tipicamente inferiore adsorbimento di proteine ​​non specifico; nessun limite di dimensione di esclusione a causa di legame complesso proteico avviene sulla superficie tallone piuttosto che all'interno i pori; e facilità di manipolazione e movimentazione con magneti.

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:

  1. Cristea, I. M., & Chait, B. T. Conjugation of magnetic beads for immunopurification of protein complexes. Cold Spring Harbor Protocols. 2011 (5) (2011).
  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).
  10. Wang, X., & Huang, L. Identifying dynamic interactors of protein complexes by quantitative mass spectrometry. Mol Cell Proteomics. 7 (1), 46–57 (2008).
  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).
  12. 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).
  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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Biochimica
cattura affinit
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immunoprecipitazione
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cryomilling
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proteine di purificazione complesso

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