Tandem Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells Using In Vivo Biotinylation

Jianlong Wang1, Alan B. Cantor1, Stuart H. Orkin2

1 Children's Hospital and the Dana Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts, 2 The Howard Hughes Medical Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1B.5
DOI:  10.1002/9780470151808.sc01b05s11
Online Posting Date:  October, 2009
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Abstract

Streptavidin affinity purification of protein complexes, in combination with in vivo biotinylation of critical transcription factors, has contributed to the analysis of the pluripotent state in mouse embryonic stem (ES) cells and made it possible to construct a protein‐protein interaction network.This has facilitated discovery of novel pluripotency factors and a better understanding of stem cell pluripotency. Here we describe detailed procedures for an in vivo biotinylation system setup in mouse ES cells, and affinity purification of multi‐protein complexes using in vivo biotinylation. In addition, we present a protocol employing SDS‐PAGE fractionation to reduce sample complexity prior to submission for mass spectrometry (MS) protein identification. Curr. Protoc. Stem Cell Biol. 11:1B.5.1‐1B.5.17. © 2009 by John Wiley & Sons, Inc.

Keywords: tandem affinity purification; in vivo biotinylation; protein‐protein interaction; embryonic stem cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Establishment of ES Cell Lines Expressing BirA and Sub‐Endogenous Biotinylated Proteins
  • Basic Protocol 2: Tandem Affinity Purification of Protein Complexes
  • Support Protocol 1: SDS‐Page Fractionation of Protein Complexes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Establishment of ES Cell Lines Expressing BirA and Sub‐Endogenous Biotinylated Proteins

  Materials
  • J1 ES cells (ATCC, cat. no. SCRC‐1010)
  • ES medium (see recipe)
  • IEF medium (see recipe)
  • 0.05% (w/v) trypsin (Mediatech, cat. no. 25‐052‐CI)
  • 0.25% (w/v) trypsin (Mediatech, cat. no. 25‐053‐CI)
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • pEF1αBirAV5‐neo plasmid (see Fig. ; available from the author upon request)
  • TE buffer (see recipe)
  • 300 µg/ml G418 (from 300 mg/ml stock; see recipe)
  • 2× freezing medium (see recipe)
  • RIPA buffer (Boston BioProducts, cat. no. BP‐115)
  • anti‐V5‐HRP (Invitrogen, cat. no. 46‐0708)
  • pEF1αFlagbiotin (FLBIO)‐puro plasmid (see Fig. ; available from the author upon request) Puromycin (see recipe)
  • Streptavidin‐HRP (Amersham, cat. no. RPN1231)
  • 6‐well, 24‐well, 48‐well, and 10‐cm IEF plates (see recipe)
  • 15‐ml conical tubes (Corning, cat. no. 430791)
  • 0.4‐cm gap cuvette for electroporator (Bio‐Rad, cat. no. 165‐2008)
  • Gene Pulsor II (electroporation; Bio‐Rad)
  • 37°C, 5% CO 2 incubator
  • U‐bottom 96‐well plate
  • 200‐µl pipettor
  • Multi‐channel pipettor (e.g., 12‐channel pipettor)
  • Parafilm
  • Gelatin‐coated cell culture plates (see recipe)

Basic Protocol 2: Tandem Affinity Purification of Protein Complexes

  Materials
  • Gelatin‐coated ES cell culture dishes (see recipe)
  • BirA‐only and BirA+ Flagbiotagging ES cells (established in protocol 1)
  • ES medium (see recipe)
  • 0.05% (w/v) trypsin (Mediatech, cat. no. 25‐052‐CI)
  • 0.25% (w/v) trypsin (Mediatech, cat. no. 25‐053‐CI)
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • Nuclear extract buffer A (see recipe)
  • Protease inhibitor cocktail (Sigma Mammalian Protease Inhibitor cocktail)
  • Trypan blue (Invitrogen, cat. no. 15250‐061)
  • Nuclear extract buffer B (see recipe)
  • Bradford assay: Protein concentration Bio‐Rad Dye kit (Bio‐Rad, cat. no. 500‐0006)
  • IP350 buffer with different NP40 concentrations (see reciperecipes)
  • Protein G–agarose (Roche, cat. no. 11‐243‐233001)
  • FLAG M2‐agarose beads (Sigma, cat. no. A2220‐5ML)
  • FLAG peptide (Sigma, F‐3290)
  • Streptavidin‐agarose beads (Invitrogen, cat. no. 15942‐050)
  • 2× SDS sample buffer (see recipe)
  • 250‐ml conical plastic bottles (Corning, cat. no. 430776)
  • Centrifuge with a JS 4.2 rotor or equivalent
  • 50‐ml conical tubes (Corning, cat. no. 430829)
  • Glass Dounce homogenizer (40‐ml size) with type B pestle (Wheaton, cat. no. 432‐1273)
  • Drawn‐out glass Pasteur pipet
  • Glass Dounce homogenizer (15‐ml size) with type B pestle (Wheaton, cat. no. 432‐1272)
  • NALGENE high‐speed centrifuge tube (cat. no. 3114‐0050)
  • Rotating wheel (Scientific Equipment Products, cat no. 60448)
  • 15‐ml conical tubes (Corning, cat. no. 430791)
  • 1.5‐ and 2‐ml screw‐cap tube
  • Additional reagents and equipment for counting cells using a hemacytometer (Phelan, ), determining cell viability using trypan blue staining (Strober, ), and determining protein concentration using the Bradford assay (Siu et al., )

Support Protocol 1: SDS‐Page Fractionation of Protein Complexes

  Materials
  • Affinity‐purified complexes with biotinylated FLAG‐tagged protein ( protocol 2)
  • 30% Acrylamide/Bis solution (37.5:1) (Bio‐Rad, cat. no. 161‐0158)
  • Colloidal Coomassie stain (Invitrogen, cat. no. 46‐7015 and 46‐7016)
  • HPLC‐grade water (American Bioanalytical)
  • YM‐10 Centricon (10,000 MWCO; Amicon Bioseparations, cat. no. 4205)
  • 37°C water bath
  • Avanti J25 centrifuge using a JA‐25.50 fixed‐angle rotor
  • 1.5‐ml microcentrifuge tubes
  • Bio‐Rad Protean II xi basic unit with casting stand (Bio‐Rad, cat. no. 165‐1834)
  • Scalpel or razor blade
  • Additional reagents and equipment for preparing a large denaturing polyacrylamide gel (Gallagher, )
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Figures

Videos

Literature Cited

Literature Cited
   Beckett, D., Kovaleva, E., and Schatz, P.J. 1999. A minimal peptide substrate in biotin holoenzyme synthetase‐catalyzed biotinylation. Protein Sci. 8:921‐929.
   Chubet, R.G. and Brizzard, B.L. 1996. Vectors for expression and secretion of FLAG epitope‐tagged proteins in mammalian cells. Biotechniques 20:136‐141.
   Conner, D.A. 2000. Mouse embryo fibroblast (MEF) feeder cell preparation. Curr. Protoc. Molec. Biol. 51:23.2.1‐23.2.7.
   Cronan, J.E., Jr. 1990. Biotination of proteins in vivo. A post‐translational modification to label, purify, and study proteins. J. Biol. Chem. 265:10327‐10333.
   de Boer, E., Rodriguez, P., Bonte, E., Krijgsveld, J., Katsantoni, E., Heck, A., Grosveld, F., and Strouboulis, J. 2003. Efficient biotinylation and single‐step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 100:7480‐7485.
   Gallagher, S. 2006. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Molec. Biol. 75:10.2A.1‐10.2A.38.
   Kim, J., Cantor, A.B., Orkin, S.H., and Wang, J. 2009. Use of in vivo biotinylation to study protein‐protein and protein‐DNA interactions in mouse embryonic stem cells. Nat. Protoc. 4:506‐517.
   Kim, J., Chu, J., Shen, X., Wang, J., and Orkin, S.H. 2008. An extended transcriptional network for pluripotency of embryonic stem cells. Cell 132:1049‐1061.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Siu, F.K.Y., Lee, L.T.O., and Chow, B.K.C. 2008. Southwestern blotting in investigating transcriptional regulation. Nat. Protoc. 3:51‐58.
   Strober, W. 1997. Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. 21:A.3B.1‐A.3B.2.
   Wang, J., Rao, S., Chu, J., Shen, X., Levasseur, D.N., Theunissen, T.W., and Orkin, S.H. 2006. A protein interaction network for pluripotency of embryonic stem cells. Nature 444:364‐368.
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