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Abstract

Biochemistry

Protein Complex Affinity Capture von Cryomilled Mammalian Cells

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 …

Affinitätseinfang ist eine wirksame Technik für die endogene Proteinkomplexe für die weitere Untersuchung zu isolieren. Wenn sie in Verbindung mit einem Antikörper verwendet wird, wird diese Technik auch als Immunpräzipitation häufig bezeichnet. Affinitätseinfangen in einer Labormaßstab und in einer Hochdurchsatz-Rahmen angewendet werden. Wenn sie mit Protein-Massenspektrometrie gekoppelt, Affinitätseinfangen hat sich als Zugpferd der Interaktom Analyse. Obwohl es möglicherweise viele Möglichkeiten gibt, beteiligt die zahlreichen Schritte auszuführen, implementieren die folgenden Protokolle unsere bevorzugte Methoden. Zwei Merkmale sind unverwechselbar: die Verwendung von cryomilled Zell Pulver Zellextrakten zu produzieren, und Antikörper-gekoppelten paramagnetischen Kügelchen als Affinitätsmedium. In vielen Fällen haben wir überlegene Ergebnisse zu denen mit konventionelleren Affinitätseinfangen Verfahren erhalten wurde. Kryomahlen vermeidet zahlreiche mit anderen Formen von Zellbruch verbundenen Probleme. Es sorgt für eine effiziente Bruch des Materials bei gleichzeitiger Vermeidung denatration Probleme mit Heizung oder Schäumen verbunden. Es behält die native Proteinkonzentration bis zu dem Punkt der Extraktion, makromolekulare Dissoziation zu mildern. Es reduziert die Zeit extrahierten Proteine ​​in Lösung zu verbringen, schädliche enzymatischen Aktivitäten zu begrenzen, und es kann die nicht-spezifische Adsorption von Proteinen durch das Affinitätsmedium zu reduzieren. Micron-Skala magnetischen Affinitätsmedien haben mehr alltäglich in den letzten Jahren geworden, immer mehr die traditionellen Agarose- ersetzt und Sepharose-basierte Medien. Primäre Vorteile von magnetischen Medien umfassen typischerweise geringere nicht-spezifische Protein-Adsorption; keine Größenausschlussgrenze, da Protein-Komplex tritt eher auf der Oberfläche der Kügelchen Bindung als in den Poren; und eine einfache Manipulation und Handhabung mit Hilfe von Magneten.

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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  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).
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  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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  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).
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  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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