UNIT19.20 Strep/FLAG Tandem Affinity Purification
(SF-TAP)to Study Protein Interactions
Christian Johannes Gloeckner,1Karsten Boldt,1,2and Marius Ueffing1,2
1Helmholtz Zentrum M¨u nchen,Neuherberg,Germany
2Technical University of Munich,Munich,Germany
ABSTRACT
In recent years,several methods have been developed to analyze protein-protein interac-
tions 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.Depend-
ing 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.C 2009by John Wiley
&Sons,Inc.
Keywords:SF-TAP r tandem affinity purification r protein complexes
INTRODUCTION
The analysis of protein-protein interactions under native conditions has been a challenge
ever since immunoprecipitation(IP)became a common methodology.Low yields and
nonspecific binding of proteins have been associated with IP.On the other hand,IP
facilitates targeted analysis of protein interactions with respect to a predefined protein
of interest,given that a suitable antibody is available that features monospecificity and
selectivity for this protein.
Tandem affinity purification(TAP;UNIT19.19)can significantly reduce the background
caused by nonspecific binding of proteins,as it combines two affinity purifications based
on two different affinity matrices(Rigaut et al.,1999).TAP has been widely used to
purify protein complexes from different species(Collins and Choudhary,2008).The TAP
technique was originally developed to analyze the yeast protein interactome(Gavin et al.,
2002).Although the original TAP tag,consisting of a Protein A-tag,a TEV(tobacco etch
virus)protease cleavage site,and a calmodulin binding peptide(CBP)tag,has already
been successfully used in mammalian cells(Bouwmeester et al.,2004),several features
of thisfirst-generation tag remain suboptimal,such as its high molecular mass(21kDa),
the dependency on proteolytic cleavage,and CBP,which may interfere with calcium
signaling within eukaryotic cells.This unit presents an alternative TAP protocol for the
isolation of protein complexes from higher eukaryotic cells.The Strep/FLAG tandem
affinity purification(SF-TAP)tag(Gloeckner et al.,2007)combines a tandem Strep-tag
II(Skerra and Schmidt,2000;Junttila et al.,2005)and a FLAG tag,resulting in a small
4.6-kDa tag.Both moieties have a medium affinity and avidity to their immobilized
binding partners.Therefore,the tagged fusion proteins and their binding partners can
be recovered under native conditions without the need for time-consuming proteolytic
cleavage.In thefirst step,desthiobiotin is used for elution of the SF-TAP fusion protein
from the Strep-Tactin matrix.In the second step,the FLAG octapeptide is used for elution
of the SF-TAP fusion protein from the anti-FLAG M2affinity matrix.An overview of the Current Protocols in Protein Science19.20.1-19.20.19,August2009
Published online August2009in Wiley Interscience(www.interscience.wiley).
DOI:10.1002/0471140864.ps1920s57
Copyright C 2009John Wiley&Sons,Inc.Identification of Protein Interactions
19.20.1 Supplement57
Strep/FLAG
Tandem Affinity
Purification (SF-TAP)
19.20.2
Supplement 57Current Protocols in Protein Science A B    1. purification    2. purification binding to Strep-Tactin binding to FLAG matrix elution with desthiobiotin elution with FLAG peptide Key:SF-TAP desthiobiotin FLAG peptide Figure 19.20.1The S trep/FLAG ta n dem affin ity p u rificatio n .(A )N-a n d C-termi n al S F-T AP ta gs (POI,protei n of i n tere s t).(B )Overview of both p u rificatio n s tep s .(
1)P u rificatio n by the ta n dem S trep-ta g II moiety:bi n di ng to S trep-T acti n matrix followed by el u tio n with de s thiobioti n .(2)P u rificatio n by the FLAG-ta g moiety:bi n di ng to a n ti-FLAG M2affin ity matrix followed by el u -tio n with FLAG peptide.Abbreviatio ns :s p.,s pecific i n teractor s (s how n a s g ray circle s );n .s p.,n o ns pecific protei ns (co n tami n a n t s ;s how n a s white circle s ).SF-TAP technique and the tag sequence is shown in Figure 19.20.1.The SF-TAP protocol represents an efficient,fast and straightforward purification of protein complexes from mammalian cells within 2hr.This unit describes the full workflow,starting with the cell culture work needed for recombinant expression of the SF-TAP fusion proteins,followed by the SF-TAP protocol (see Basic Protocol 1)and ending with mass spectrometric analysis of the samples (see Basic Protocol 4).Special focus is given to the crucial step of sample preparation for mass spectrometry.For the identification of associated proteins following SF-TAP,the volume of the SF-TAP eluates is reduced by ultrafiltration using centrifugal units with a low molecular weight cut-off or by chloroform/methanol precipitation (see Support Protocol 2).The samples are then directly subjected to proteolytic digestion (see Basic Protocol 2)for analysis on a nano liquid chromatography (LC)–coupled electron spray
Identification of Protein Interactions 19.20.3Current Protocols in Protein Science Supplement 57
Figure 19.20.2Flow chart of a S F-T AP approach i n cl u di ng M S ide n tificatio n of cop u rified pro-tei ns .Thi s figu re co nn ect s all protocol s pre s e n ted i n thi s un it.
tandem mass spectrometer.For complex samples,which contain many proteins,an alternative procedure for SDS-PAGE pre-fractionation is provided,including a method for sensitive MS-compatible Coomassie protein staining (see Support Protocol 3)followed by in-gel proteolytic digestion (see Basic Protocol 3).By reducing sample complexity,pre-fractionation helps to increase the number of protein identifications on state-of-the-art LC-coupled tandem mass spectrometers.Representative MS-analysis protocols are provided for an Orbitrap mass spectrometer (Thermo Fisher Scientific),a fast and sensitive system allowing high identification rates from SF-TAP purifications even with low amounts of protein in the sample (see Basic Protocol 4).Finally,a strategy for meta analysis of mass spectrometric data sets using the Scaffold software is provided (see Support Protocol 4).It can generally be used for the analysis of large MS/MS data sets.
Figure 19.20.2provides a flowchart of the entire analytical process.
Strep/FLAG
Tandem Affinity
Purification (SF-TAP)
19.20.4
Supplement 57Current Protocols in Protein Science
BASIC
PROTOCOL 1
STREP/FLAG TANDEM AFFINITY PURIFICATION (SF-TAP)OF PROTEIN COMPLEXES FROM HEK293CELLS A flowchart of the SF-TAP procedure is shown in Figure 19.20.3.Materials HEK293cells (ATCC no.CRL-1573)Complete DMEM containing 10%FBS (APPENDIX 3C )SF-TAP vectors with appropriate insert,and empty control plasmid (see Critical Parameters)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 2-1206-10)Tris-buffered saline (TBS;see recipe)Wash buffer (see recipe)Desthiobiotin elution buffer:dilute 10×buffer E (IBA 2-1000-025)1:10in H 2O (final concentration,2mM desthiobiotin)Anti–FLAG M2agarose (Sigma-Aldrich)FLAG elution buffer (see recipe)14-cm tissue cu
lture plates Cell scraper Millex GP 0.22-μm syringe-driven filter units (Millipore)End-over-end rotator Microspin columns (GE 27-3565-01)End-over-end rotator Microcon YM-3centrifugal filter devices (Millipore)Additional reagents and equipment for transfection of mammalian cells (UNIT 5.10)Transfect HEK293cells 1.Seed HEK293cells on 14-cm plates at ∼1–2×107cells per dish in complete DMEM medium containing 10%FBS.The amount of cells used for SF-TAP purification can be varied depending on the ex-pression levels of the bait protein.Usually,four 14-cm dishes,corresponding to a final amount of ∼4×108HEK293cells,is a good starting point.Strong overexpression of the bait protein usually increases copurification of heat-shock proteins such as HSP70.For in-depth analysis,it is therefore recommended to generate cell lines stably expressing the bait protein.See Support Protocol 1for a stable transfection method.2.Grow cells overnight.3.Transfect cells with the SF-TAP plasmids using a transfection reagent of choice (according to manufacturer’s protocols).HEK293cells can be easily transfected with lipophilic transfection reagents.The trans-fection efficiency is usually >80%.For a typical SF-TAP experiment,1to 4μg plasmid per 14-cm dish is used.Depending on the cell type other transfection reagents may be favorable (also see UNIT 5.10).Although SF-TAP purifications typically exhibit low background caused by nonspecific binding of proteins to the affinity matrix,a suitable negative control should be used in every experiment.Cells transfected with the empty expression vectors may be use
d in the same amount as for the SF-TAP-tagged bait protein.However,the tag is quite small and expressed at low levels if not fused to a protein.Thus,the untransfected cell line is an acceptable,simple,and inexpensive alternative for a negative control.
Identification of Protein Interactions 19.20.5Current Protocols in Protein Science Supplement 571-4 × 108 HEK293 cell s
(1-4 co n fl u e n t 14-cm plate s )
expre ss i ng  S F-TAP f us io n  protei n
ly s i s
(15 mi n  4
C)vol u me
red u ctio n
ce n trif ug atio n (10 mi n  10,000 × g )
a n aly s i s
retai n su per n ata n t fi n al
el u ate
i n c u batio n  with
50 μl/plate S trep-Tacti n matrix (1 hr)el u tio n  with
200 μl FLAG
el u tio n  b u ffer
(10 mi n )
wa s h 3 time s  with 500 μl wa s h b u ffer (s pi n  5 s ec, 100 × g )wa s h 3 time s  with
500 μl wa s h b u ffer
(s pi n  5 s ec, 100 × g )
el u tio n  with 500 μl de s thiobioti n el u tio n  b u ffer (10 mi n )i n c u batio n  with
25 μl/plate
a n ti-FLAG M2
a g aro s e
(1 hr)
Figure 19.20.3Flow chart for the S F-T AP proced u re.
4.Let cells grow for 48hr.
If necessary,cells can be starved in DMEM without FBS for 12hr prior to harvesting.Starving might be desirable if cell signaling is to be analyzed,especially prior to differ-ential treatment with growth factors,to eliminate effects of serum growth factors.Lyse cells
5.Remove medium from the plates.
6.Optional:Rinse cells in warm PBS.
Strep/FLAG
Tandem Affinity Purification (SF-TAP)19.20.6
scraperSupplement 57Current Protocols in Protein Science 7.Scrape off cells in 1ml lysis buffer per 14-cm plate on ice using a cell scraper,and combine lysates from each experimental condition in a 1.5-ml microcentrifuge tube.8.Lyse cells by incubating 15min on ice with mixing by hand from time to time.9.Pellet cell debris,including nuclei,by centrifuging 10min at 10,000×g ,4◦C.10.Clear lysate supernatant by filtration through a 0.22-μm syringe filter.Perform SF-TAP 11.Wash Strep-Tactin Superflow resin twice,each time with 4resin volumes TBS and once with 4resin volumes lysis buffer.12.Incubate lysates with 50μl per 14-cm plate of settled Strep-Tactin Superflow resin for 1hr at 4◦C (use an end-over-end rotator to keep the resin evenly distributed).Note that a maximum of 200μl settled resin per spin column should not be exceeded.If more than four 14-cm plates (∼4×108HEK293cells)are used,reduce the volume per plate or use additional spin columns in step 13.13.Centrifuge for 30sec at 7000×g ,4◦C,remove the supernatant until 500μl remains,and transfer resin to a microspin column.Snap off bottom closure of the spin column prior to use.The maximum volume of the spin columns is 650μl.Alternatively,centrifugations for wash and elution steps can be performed at room temperature if no cooled centrifuge is available.14.Remove remaining supernatan
t by centrifugation in the spin column for 5sec at 100×g ,then wash resin three times,each time with 500μl wash buffer (centrifuge 5sec at 100×g each time to remove the supernatant)at 4◦C.Replug spin columns with inverted bottom closure prior to adding the elution buffer in step 15.IMPORTANT NOTE:Do not allow the resin to run dry.Depending on the bait protein,this markedly reduces the yield.15.Add 500μl desthiobiotin elution buffer and gently mix the resin by hand for 10min on ice.16.Remove the plug of the spin column,transfer the column to a new collection tube,and collect the eluate by centrifuging 10sec at 2000×g ,4◦C.If spin columns were closed by the top screw cap during incubation with elution buffer,the cap needs to be removed prior to centrifugation,to allow the pressure to balance out.17.Wash anti–FLAG M2agarose resin three times,each time with 4resin volumes TBS.Suspend resin in TBS and transfer it to microspin columns,then remove the buffer by centrifuging 5sec at 100×g .25μl settled resin per 14-cm plate will be needed.18.Transfer eluate from step 16corresponding to each 14-cm plate to a microspin column containing 25μl settled anti-FLAG M2agarose prepared as in step 17.19.Plug columns,close columns with top screw caps,and incubate for 1hr at 4◦C (on an end-over-end rotator).20.Wash once with 500μl wash buffer,and then twice,each time with 500μl TBS (centrifuge 5sec at 100×g each time to remove the supernatant)at 4◦C.21.For elution,incubate with 4bead volumes (at least 200μl)FLAG elution buffer for 10min,keeping the columns plugged and gently mixing the resin several times.22.After incubation,re
move the plugs and top screws of the spin columns,transfer to new collection tubes,and collect the eluate(s)by centrifugation (10sec at 2000×g ).

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