Strep/FLAG Tandem Affinity Purification (SF‐TAP) to Study Protein Interactions

Christian Johannes Gloeckner1, Karsten Boldt2, Marius Ueffing2

1 Helmholtz Zentrum München, Neuherberg, Germany, 2 Technical University of Munich, Munich, Germany
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.20
DOI:  10.1002/0471140864.ps1920s57
Online Posting Date:  August, 2009
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Abstract

In recent years, several methods have been developed to analyze protein‐protein interactions under native conditions. One of them, tandem affinity purification (TAP), combines two affinity‐purification steps to allow isolation of high‐purity protein complexes. This unit presents a methodological workflow based on an SF‐TAP tag comprising a doublet Strep‐tag II and a FLAG moiety optimized for rapid as well as efficient tandem affinity purification of native proteins and protein complexes in higher eukaryotic cells. Depending on the stringency of purification conditions, SF‐TAP allows both the isolation of a single tagged‐fusion protein of interest and purification of protein complexes under native conditions. Curr. Protoc. Protein Sci. 57:19.20.1‐19.20.19. © 2009 by John Wiley & Sons, Inc.

Keywords: SF‐TAP; tandem affinity purification; protein complexes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Strep/Flag Tandem Affinity Purification (SF‐TAP) of Protein Complexes From HEK293 Cells
  • Support Protocol 1: Generation of HEK293 Clones Stably Expressing SF‐TAP‐Tagged Proteins
  • Support Protocol 2: Chloroform/Methanol Precipitation of Proteins
  • Basic Protocol 2: In‐Solution Digest of Proteins for Mass Spectrometric Analysis
  • Basic Protocol 3: Pre‐Fractionation Via SDS‐Page and In‐Gel Digestion Prior to LC‐MS/MS Analysis
  • Support Protocol 3: Quick MS‐Compatible Colloidal Coomassie Stain of Proteins After SDS‐PAGE Separation
  • Basic Protocol 4: LC‐MS/MS Analysis of Digested SF‐TAP Samples
  • Support Protocol 4: Database Search and Data Analysis by Scaffold
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Strep/Flag Tandem Affinity Purification (SF‐TAP) of Protein Complexes From HEK293 Cells

  Materials
  • HEK293 cells (ATCC no. CRL‐1573)
  • Complete DMEM containing 10% FBS ( appendix 3C)
  • SF‐TAP vectors with appropriate insert, and empty control plasmid (see )
  • Negative control (see annotation to step 3, below)
  • Transfection reagent of choice (see unit 5.10)
  • Phosphate‐buffered saline (PBS; appendix 2E), prewarmed
  • Lysis buffer (see recipe)
  • Strep‐Tactin Superflow resin (IBA GmbH, cat. no. 2‐1206‐10)
  • Tris‐buffered saline (TBS; see recipe)
  • Wash buffer (see recipe)
  • Desthiobiotin elution buffer: dilute 10× buffer E (IBA GmbH, cat. no. 2‐1000‐025) 1:10 in H 2O (final concentration, 2 mM desthiobiotin)
  • Anti–FLAG M2 agarose (Sigma‐Aldrich)
  • FLAG elution buffer (see recipe)
  • 14‐cm tissue culture plates
  • Cell scraper
  • Millex GP 0.22‐µm syringe‐driven filter units (Millipore)
  • End‐over‐end rotator
  • Microspin columns (GE Healthcare, cat. no. 27‐3565‐01)
  • End‐over‐end rotator
  • Microcon YM‐3 centrifugal filter devices (Millipore)
  • Additional reagents and equipment for transfection of mammalian cells (unit 5.10)

Support Protocol 1: Generation of HEK293 Clones Stably Expressing SF‐TAP‐Tagged Proteins

  Materials
  • HEK293 cells (ATCC no. CRL‐1573)
  • Complete DMEM containing 10% FBS ( appendix 3C)
  • SF‐TAP vectors with appropriate insert, and empty control plasmid (see )
  • Transfection reagent of choice (see unit 5.10)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Complete DMEM medium ( appendix 3C)
  • G418 (PAA Laboratories, http://www.paa.com)
  • Freezing solution: 90% fetal bovine serum (FBS; Invitrogen)/10% dimethylsulfoxide (DMSO; AR grade)
  • Lysis buffer (see recipe)
  • Blocking reagent: 5% (w/v) nonfat dry milk in TBS (see recipe for TBS) containing 0.1% (v/v) Tween 20
  • Anti‐FLAG M2 antibody (Sigma‐Aldrich)
  • 10‐cm tissue culture dishes
  • 12‐well and 6‐welll tissue culture plates
  • Centrifuge
  • 2‐ml cryovials (Nunc)
  • Additional reagents and equipment for transfection of mammalian cells (unit 5.10), trypsinization and counting of cells (unit 5.10), and immunoblotting (unit 10.10)

Support Protocol 2: Chloroform/Methanol Precipitation of Proteins

  Materials
  • SF‐TAP eluate (from protocol 1)
  • Methanol (AR grade)
  • Chloroform (AR grade)
  • 2‐ml polypropylene sample tubes

Basic Protocol 2: In‐Solution Digest of Proteins for Mass Spectrometric Analysis

  Materials
  • Precipitated protein (see protocol 3)
  • 50 mM ammonium bicarbonate (freshly prepared)
  • RapiGest SF (Waters): prepare 2% (10×) stock solution in deionized water
  • 100 mM DTT (prepare from 500 mM stock solution; store stock up to 6 months at −20°C)
  • 300 mM iodoacetamide (prepare fresh)
  • 50× (0.5 µg/µl) trypsin stock solution (Promega; store at −20°C)
  • Concentrated (37%) HCl
  • 60°C incubator
  • Polypropylene inserts (Supelco, cat. no. 24722)
  • 1 to 200 µl gel‐loader pipet tips (Sorenson Bioscience, http://www.sorbio.com/contact.cfm)

Basic Protocol 3: Pre‐Fractionation Via SDS‐Page and In‐Gel Digestion Prior to LC‐MS/MS Analysis

  Materials
  • Protein sample (e.g., from protocol 1 or protocol 3)
  • 10% NuPAGE gels (Invitrogen)
  • MOPS running buffer (Invitrogen)
  • 40% and 100% acetonitrile (AR grade; prepare fresh)
  • 5 mM DTT (prepare from 500 mM stock; store stock up to 6 months at −20°C)
  • 25 mM iodoacetamide (prepare fresh)
  • Digestion solution: dilute 50× trypsin stock solution (0.5 µg/µl, Promega) 1:50 in 50 mM ammonium bicarbonate (freshly prepared)
  • 1% and 0.5% (v/v) trifluoroacetic acid (TFA; prepare fresh from 10% v/v stock)
  • 50% (v/v) acetonitrile/0.5% (v/v) TFA (prepare fresh)
  • 99.5% (v/v) acetonitrile/0.5% (v/v) TFA (prepare fresh)
  • 2% (v/v) acetonitrile/0.5% (v/v) TFA
  • Concentration units (e.g., Microcon from Millipore)
  • Scalpel
  • Polypropylene 96‐well microtiter plate: polystyrene material should be avoided since, depending on the product, polymers can be extracted from plastics which produce strong background signals in mass spectrometry
  • 60°C incubator or heating block
  • Polypropylene 0.5‐ml reaction tubes
  • Microtiter plate shaker (e.g., Vortex mixer equipped with microtiter‐plate adaptor)
  • HPLC sample tubes
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and colloidal Coomassie blue staining of gels ( protocol 6)

Support Protocol 3: Quick MS‐Compatible Colloidal Coomassie Stain of Proteins After SDS‐PAGE Separation

  Materials
  • Electrophoresed SDS gel containing protein samples of interest (e.g., from protocol 5)
  • Colloidal Coomassie staining solution (see recipe)
  • Destaining solution: 10% (v/v) ethanol/2% (v/v) orthophosphoric acid
  • Gel staining trays of appropriate size

Basic Protocol 4: LC‐MS/MS Analysis of Digested SF‐TAP Samples

  Materials
  • Digested protein sample, either from in‐solution digest ( protocol 4) or in‐gel digest ( protocol 5)
  • Nano HPLC loading buffer: 0.1% formic acid in HPLC‐grade water
  • Nano HPLC buffer A: 2% acetonitrile/0.1% formic acid in HPLC‐grade water
  • Nano HPLC buffer B: 80% acetonitrile/0.1% formic acid in HPLC‐grade water
  • HPLC vials (Dionex)
  • Nano HPLC system (UltiMate 3000, Dionex) equipped with a trap column (100 µm i.d. × 2 cm, packed with Acclaim PepMap100 C18 resin, 5 µm, 100 Å; Dionex) and an analytical column (75 µm i.d. × 15 cm, packed with Acclaim PepMap100 C18 resin, 3 µm, 100Å; Dionex)
  • Mass spectrometer: Oritrap XL with a nanospray ion source (ThermoFisher Scientific; also see unit 16.11)
  • Additional reagents and equipment for mass spectrometry (unit 16.11) and analysis of mass spectra ( protocol 8)

Support Protocol 4: Database Search and Data Analysis by Scaffold

  Materials
  • Bioworks Browser including Sequest (ThermoFisher Scientific)
  • Sequest (ThermoFisher Scientific)
  • Mascot (Matrix Science)
  • Uniref100 database (http://www.ebi.ac.uk/uniref/)
  • Scaffold (Proteome Software; http://www.proteomesoftware.com/)
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Figures

Videos

Literature Cited

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