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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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% CO₂ 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

Figure 1.B0.1 Establishment of a biotinylation system in J1 ESCs. A stable ESC line expressing the bacterial BirA enzyme was first established by transfection with a BirA‐expressing plasmid bearing the neomycin resistance ( neo ^r ) gene and G418 selection. A second plasmid containing cDNA encoding a transcription factor (TF) of interest with an N‐terminal Flag‐biotin dual tag (FLBIO) and a puromycin resistance ( puro ^r ) gene was introduced, and cells were selected with puromycin. The resulting stable line is resistant to both G418 and puromycin, and expresses FLAG‐tagged, biotinylated TF (^FLBIO TF) that can be immunoprecipitated by anti‐FLAG and streptavidin antibodies/beads.

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Figure 1.B0.2 A summary of the procedure for tandem affinity purification of multiprotein complexes in mouse ESCs. Following establishment of BirA‐only and BirA+^FLBIO TF‐expressing ES cell lines, immunoprecipitation is performed using anti‐FLAG M2 agarose (FLAG‐IP). The bound material is eluted with FLAG peptide and further purified by streptavidin affinity capture. The purified protein complexes are fractionated on SDS‐PAGE, and subjected to LC‐MS/MS to identify components of the protein complexes.

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Figure 1.B0.3 Examples of western blot analyses. (A ) A western blot with anti‐V5‐HRP to detect BirAV5 expression. (B ) A western blot using native antibody against the TF of interest to detect both endogenous and biotinylated TF proteins. The sub‐endogenous level of the biotinylated transcription factor (^FLBIO TF) and the expression level of the endogenous protein (^end TF) are indicated. NS denotes nonspecific signals. Panel B is reprinted from Kim et al. () with permission of Nature Protocols

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Literature Cited

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