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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 H₂ O (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

Figure 19.20.1 The Strep/FLAG tandem affinity purification. (A ) N‐ and C‐terminal SF‐TAP tags (POI, protein of interest). (B ) Overview of both purification steps. (1) Purification by the tandem Strep‐tag II moiety: binding to Strep‐Tactin matrix followed by elution with desthiobiotin. (2) Purification by the FLAG‐tag moiety: binding to anti‐FLAG M2 affinity matrix followed by elution with FLAG peptide. Abbreviations: sp., specific interactors (shown as gray circles); n.sp., nonspecific proteins (contaminants; shown as white circles).

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Figure 19.20.2 Flow chart of a SF‐TAP approach including MS identification of copurified proteins. This figure connects all protocols presented in this unit.

View Image

Figure 19.20.3 Flow chart for the SF‐TAP procedure.

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Videos

Literature Cited

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited