Site‐Specific Protein Labeling with SNAP‐Tags

Nelson B. Cole1

1 Laboratory of Cell Biology, National Heart, Lung, and Blood Institute/National Institutes of Health (NHLBI/NIH), Bethesda, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 30.1
DOI:  10.1002/0471140864.ps3001s73
Online Posting Date:  September, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

ABSTRACT

Site‐specific labeling of cellular proteins with chemical probes is a powerful tool for studying protein function in living cells. A number of small peptide and protein tags have been developed that can be labeled with synthetic probes with high efficiencies and specificities and provide flexibility not available with fluorescent proteins. The SNAP‐tag is a modified form of the DNA repair enzyme human O6‐alkylguanine‐DNA‐alkyltransferase, and undergoes a self‐labeling reaction to form a covalent bond with O6‐benzylguanine (BG) derivatives. BG can be modified with a wide variety of fluorophores and other reporter compounds, generally without affecting the reaction with the SNAP‐tag. In this unit, basic strategies for labeling SNAP‐tag fusion proteins, both for live cell imaging and for in vitro analysis, are described. This includes a description of a releasable SNAP‐tag probe that allows the user to chemically cleave the fluorophore from the labeled SNAP‐tag fusion. In vitro labeling of purified SNAP‐tag fusions is briefly described. Curr. Protoc. Protein Sci. 73:30.1.1‐30.1.16. © 2013 by John Wiley & Sons, Inc.

Keywords: SNAP‐tag; chemical labeling; cell biology; endocytosis; imaging

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Site‐Specific Labeling of Cell Surface SNAP‐Tag Fusion Proteins
  • Alternate Protocol 1: Site‐Specific Labeling of Intracellular SNAP‐Tag Fusion Proteins
  • Support Protocol 1: Releasable SNAP‐Tag Probe for Studying Endocytosis and Recycling
  • Basic Protocol 2: Pulse‐Chase Labeling of SNAP‐Tag Fusions Detected by In‐Gel Fluorescence
  • Alternate Protocol 2: Labeling Cell Surface Proteins with Cell‐Impermeable BG‐PEG‐Biotin Probes
  • Support Protocol 2: Synthesis of BG‐800 Substrate
  • Basic Protocol 3: In Vitro Labeling of SNAP‐Tag Fusions
  • Support Protocol 3: Labeling of SNAP‐Tag Proteins from Cell Lysates
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Site‐Specific Labeling of Cell Surface SNAP‐Tag Fusion Proteins

  Materials
  • Target cells (any adherent or suspension cell line that can express proteins from transfected DNAs may be used)
  • Culture medium (the presence of serum does not inhibit the SNAP‐tag reaction)
  • Plasmid encoding SNAP‐tag fusion protein: the original SNAP‐tag, SNAP26m, has been replaced by a faster labeling variant, SNAP f (available from NEB) that allows for reduction of substrate concentration and incubation times
  • Transfection reagent (Fugene, Roche; Lipofectamine, Invitrogen); an electroporation system (Amaxa) may also be used
  • Cell‐impermeable BG‐labeled probe (“Cell Surface” probes from NEB or custom‐synthesized; see protocol 3)
  • Hanks’ buffered salt solution (HBSS, with CaCl 2 and MgCl 2; e.g., Invitrogen) or Dulbecco's phosphate‐buffered saline (DPBS, with CaCl 2 and MgCl 2; e.g., Invitrogen)
  • Non‐fluorescent SNAP‐cell blocking reagents: SNAP Cell Block (NEB, cat. no. S9106, cell permeable), SNAP‐Surface Block (NEB, cat. no. S9143, cell impermeable), BG‐NH 2 (Toronto Research Chemicals, cat. no. A614425, cell permeable)
  • Fixative: 2% to 3.7% formaldehyde in DPBS or 100% methanol
  • Mounting medium (Fluoromount‐G, Southern Biotechnologies, etc.)
  • Glass coverslips (Daigger, Fisher, etc.) or chambered coverslips (Lab‐Tek II Chambered Coverglass, Nunc)
  • Glass microscope slides
  • Fluorescence microscope with appropriate filter set

Alternate Protocol 1: Site‐Specific Labeling of Intracellular SNAP‐Tag Fusion Proteins

  Additional Materials (also see protocol 1)
  • Cell‐permeable BG probe (e.g., SNAP‐Cell TMR Star, NEB cat. no. S9105S); other probes from NEB include SNAP‐Cell 505, SNAP‐Cell Oregon Green, and SNAP‐Cell Fluorescein

Support Protocol 1: Releasable SNAP‐Tag Probe for Studying Endocytosis and Recycling

  Additional Materials (also see protocol 1)
  • Cell‐impermeable BG‐S‐S‐Alexa Fluor 488 (for probe synthesis, see Cole and Donaldson, )
  • TCEP (Sigma or Thermo Scientific; a 500 mM stock is prepared in water, then adjusted to pH ∼7 with NaOH)

Basic Protocol 2: Pulse‐Chase Labeling of SNAP‐Tag Fusions Detected by In‐Gel Fluorescence

  Materials
  • BG‐800 [synthesized from BG‐NH 2 (Toronto Research Chemicals, cat no. A614425; http://www.trc‐canada.com/) and IRDye 800CW NHS (LI‐COR Biosciences, http://www.licor.com/); see protocol 6]
  • Unlabeled BG‐NH 2 (Toronto Research Chemicals, http://www.trc‐canada.com/)
  • Dulbecco's phosphate‐buffered saline (DPBS; with CaCl 2 and MgCl 2; e.g., Invitrogen)
  • Cell lysis buffer: 50 mM Tris·Cl, pH 7.4 ( appendix 2E)/150 mM NaCl/1% (v/v) Triton X‐100/1× protease inhibitors (complete tablets, Roche)/20 µM unlabeled BG‐NH 2
  • Coomassie blue R250 (Sigma; optional)
  • 12‐ or 24‐well tissue culture–treated plates (e.g., Falcon; Costar)
  • Cell scrapers
  • Polyacrylamide gels (e.g., Novex Tris‐glycine, Invitrogen)
  • 95°C heat block or water bath
  • Infrared fluorescence scanner (Odyssey, LI‐COR Biosciences, http://www.licor.com/): in this protocol, detection of IRDye 800CW is with an Odyssey infrared scanner; depending on the fluorophore used, gels can be scanned using a number of commercially available imagers (e.g., Typhoon from GE Healthcare or Pharos FX from BioRad) using appropriate lasers and filter sets
  • Additional reagents and equipment for site‐specific labeling of cell surface SNAP‐tag fusion proteins ( protocol 1), SDS‐PAGE (unit 10.1), and fixation and staining of gels (unit 10.1)

Alternate Protocol 2: Labeling Cell Surface Proteins with Cell‐Impermeable BG‐PEG‐Biotin Probes

  Materials
  • BG‐NH 2 (Toronto Research Chemicals, http://www.trc‐canada.com/)
  • EZ‐Link NHS‐PEG 12‐Biotin (Thermo Scientific)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Cell lysis buffer: 50 mM Tris·Cl, pH 7.4 ( appendix 2E)/150 mM NaCl/1% (v/v) Triton X‐100, containing 1× protease inhibitors (complete tablets, Roche) and 20 µM unlabeled BG‐NH 2
  • Streptavidin agarose resin (Thermo Scientific)
  • Blocking buffer (LI‐COR, http://www.licor.com) or other western blocking buffer
  • PBS‐T: PBS ( appendix 2E) containing 0.1% (v/v) Tween 20)
  • Primary antibodies to detect interacting proteins
  • 700‐nm conjugated species‐specific secondary antibodies (Invitrogen or Li‐COR, http://www.licor.com)
  • NeutrAvidin, DyLight 800 conjugate (Thermo Scientific)
  • 10‐cm dishes or 6‐well plates (Falcon, Costar, etc.)
  • End‐over‐end rotator
  • Infrared fluorescence scanner (Odyssey, LI‐COR Biosciences, http://www.licor.com/)
  • Additional reagents and equipment for reaction of BG‐NH2 with NHS esters ( protocol 6), transfection with SNAP‐tag fusion constructs ( protocol 1), SDS‐PAGE (unit 10.1), and blotting (unit 10.7),

Support Protocol 2: Synthesis of BG‐800 Substrate

  Materials
  • BG‐NH 2 (O6‐[4‐(aminomethyl)benzyl]guanine; Toronto Research Chemicals, cat. no. A614425; NEB, cat. no. S9148S)
  • Anhydrous N,N‐dimethyl formamide (DMF, Sigma‐Aldrich)
  • IRDye 800CW N‐hydroxysuccinimide (NHS) ester (Licor, cat. no. 929–70020; http://www.licor.com)
  • Triethylamine (TEA, Sigma)
  • Additional reagents and equipment for LC‐MS (unit 16.1), labeling of cells expressing SNAP‐tag fusions ( protocol 4), and SDS‐PAGE (unit 10.1)

Basic Protocol 3: In Vitro Labeling of SNAP‐Tag Fusions

  Materials
  • Purified SNAP‐tag or SNAP‐tag fusion proteins (see above)
  • Fluorophore‐labeled BG‐substrate (e.g., SNAP Vista Green; NEB, cat. no. S9147)
  • Reaction buffer (PBS) or 50 mM Tris·Cl, pH 7.5 ( appendix 2E)/100 mM NaCl/ 0.1% Tween 20
  • Dithiothreitol (DTT)
  • Infrared fluorescence scanner (Odyssey, LI‐COR Biosciences, http://www.licor.com/)

Support Protocol 3: Labeling of SNAP‐Tag Proteins from Cell Lysates

  Materials
  • Cells expressing SNAP‐tag fusion protein (see protocol 4, step 1)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • 0.25% trypsin (Lonza)
  • Lysis buffer: 50 mM Tris·Cl, pH 7.5 ( appendix 2E)/100 mM NaCl/0.1% Tween 20 or 50 mM Tris·Cl, pH 7.4 ( appendix 2E)/150 mM NaCl/1% (v/v) Triton X‐100; just before use add DTT to 1 to 5 mM and an EDTA‐free protease inhibitor cocktail (e.g., Complete from Roche or CelLytic M from Sigma) to 1×
  • Tissue culture‐treated dishes (35 or 60 mm) or multi‐well plates (6 or 12 well) (Falcon; Costar, etc.)
  • Cell lifter (Corning)
  • Infrared fluorescence scanner (Odyssey, LI‐COR Biosciences, http://www.licor.com/)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

  Banala, S., Maurel, D., Manley, S., and Johnsson, K. 2012. A caged, localizable rhodamine derivative for superresolution microscopy. ACS Chem. Biol. 7:289–293.
  Cai, Y., Rossier, O., Gauthier, N.C., Biais, N., Fardin, M.A., Zhang, X., Miller, L.W., Ladoux, B., Cornish, V.W., and Sheetz, M.P. 2010. Cytoskeletal coherence requires myosin‐IIA contractility. J. Cell Sci. 123:413–423.
  Chen, I., Howarth, M., Lin, W., and Ting, A.Y. 2005. Site‐specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat. Methods 2:99–104.
  Cole, N.B. and Donaldson, J.G. 2012. Releasable SNAP‐tag probes for studying endocytosis and recycling. ACS Chem. Biol. 7:464–469.
  Comps‐Agrar, L., Maurel, D., Rondard, P., Pin, J.P., Trinquet, E., and Prezeau, L. 2011. Cell‐surface protein‐protein interaction analysis with time‐resolved FRET and snap‐tag technologies: Application to G protein‐coupled receptor oligomerization. Methods Mol. Biol. 756:201–214.
  Eyster, C.A., Higginson, J.D., Huebner, R., Porat‐Shliom, N., Weigert, R., Wu, W.W., Shen, R.F., and Donaldson, J.G. 2009. Discovery of new cargo proteins that enter cells through clathrin‐independent endocytosis. Traffic 10:590–599.
  Gautier, A., Juillerat, A., Heinis, C., Correa, I.R., Jr., Kindermann, M., Beaufils, F., and Johnsson, K. 2008. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15:128–136.
  George, N., Pick, H., Vogel, H., Johnsson, N., and Johnsson, K. 2004. Specific labeling of cell surface proteins with chemically diverse compounds. J. Am. Chem. Soc. 126:8896–8897.
  Griffin, B.A., Adams, S.R., and Tsien, R.Y. 1998. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281:269–272.
  Hinner, M.J. and Johnsson, K. 2010. How to obtain labeled proteins and what to do with them. Curr. Opin. Biotechnol. 21:766–776.
  Jing, C. and Cornish, V.W. 2011. Chemical tags for labeling proteins inside living cells. Acc. Chem. Res. 44:784–792.
  Kamiya, M. and Johnsson, K. 2010. Localizable and highly sensitive calcium indicator based on a BODIPY fluorophore. Anal. Chem. 82:6472–6479.
  Keppler, A. and Ellenberg, J. 2009. Chromophore‐assisted laser inactivation of alpha‐ and gamma‐tubulin SNAP‐tag fusion proteins inside living cells. ACS Chem. Biol. 4:127–138.
  Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H., Vogel, H., and Johnsson, K. 2003. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21:86–89.
  Keppler, A., Kindermann, M., Gendreizig, S., Pick, H., Vogel, H., and Johnsson, K. 2004a. Labeling of fusion proteins of O6‐alkylguanine‐DNA alkyltransferase with small molecules in vivo and in vitro. Methods 32:437–444.
  Keppler, A., Pick, H., Arrivoli, C., Vogel, H., and Johnsson, K. 2004b. Labeling of fusion proteins with synthetic fluorophores in live cells. Proc. Natl. Acad. Sci. U.S.A. 101:9955–9959.
  Keppler, A., Arrivoli, C., Sironi, L., and Ellenberg, J. 2006. Fluorophores for live cell imaging of AGT fusion proteins across the visible spectrum. Biotechniques 41:167#x02013;170, 172, 174–165.
  Klein, T., Loschberger, A., Proppert, S., Wolter, S., van de Linde, S., and Sauer, M. 2011. Live‐cell dSTORM with SNAP‐tag fusion proteins. Nat. Methods 8:7–9.
  Kobayashi, T., Komatsu, T., Kamiya, M., Campos, C., Gonzalez‐Gaitan, M., Terai, T., Hanaoka, K., Nagano, T., and Urano, Y. 2012. Highly activatable and environment‐insensitive optical highlighters for selective spatiotemporal imaging of target proteins. J. Am. Chem. Soc. 134:11153–11160.
  Komatsu, T., Johnsson, K., Okuno, H., Bito, H., Inoue, T., Nagano, T., and Urano, Y. 2011. Real‐time measurements of protein dynamics using fluorescence activation‐coupled protein labeling method. J. Am. Chem. Soc. 133:6745–6751.
  Lin, M.Z. and Wang, L. 2008. Selective labeling of proteins with chemical probes in living cells. Physiology 23:131–141.
  Los, G.V., Encell, L.P., McDougall, M.G., Hartzell, D.D., Karassina, N., Zimprich, C., Wood, M.G., Learish, R., Ohana, R.F., Urh, M., Simpson, D., Mendez, J., Zimmerman, K., Otto, P., Vidugiris, G., Zhu, J., Darzins, A., Klaubert, D.H., Bulleit, R.F., and Wood, K.V. 2008. HaloTag: A novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3:373–382.
  Luo, S., Wehr, N.B., and Levine, R.L. 2006. Quantitation of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast Green. Anal. Biochem. 350:233–238.
  Maurel, D., Comps‐Agrar, L., Brock, C., Rives, M.L., Bourrier, E., Ayoub, M.A., Bazin, H., Tinel, N., Durroux, T., Prézeau, L., Trinquet, E., and Pin, J.P. 2008. Cell‐surface protein‐protein interaction analysis with time‐resolved FRET and snap‐tag technologies: Application to GPCR oligomerization. Nat. Methods 5:561–567.
  Maurel, D., Banala, S., Laroche, T., and Johnsson, K. 2010. Photoactivatable and photoconvertible fluorescent probes for protein labeling. ACS Chem. Biol. 5:507–516.
  Mollwitz, B., Brunk, E., Schmitt, S., Pojer, F., Bannwarth, M., Schiltz, M., Rothlisberger, U., and Johnsson, K. 2012. Directed evolution of the suicide protein O(6)‐alkylguanine‐DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability. Biochemistry 51:986–994.
  O'Hare, H.M., Johnsson, K., and Gautier, A. 2007. Chemical probes shed light on protein function. Curr. Opin. Struct. Biol. 17:488–494.
  Srikun, D., Albers, A.E., Nam, C.I., Iavarone, A.T., and Chang, C.J. 2010. Organelle‐targetable fluorescent probes for imaging hydrogen peroxide in living cells via SNAP‐Tag protein labeling. J. Am. Chem. Soc. 132:4455–4465.
  Sun, X., Zhang, A., Baker, B., Sun, L., Howard, A., Buswell, J., Maurel, D., Masharina, A., Johnsson, K., Noren, C.J., Xu, M.Q., and Corrêa, I.R. Jr. 2011. Development of SNAP‐tag fluorogenic probes for wash‐free fluorescence imaging. Chembiochem 12:2217–2226.
  Tomat, E., Nolan, E.M., Jaworski, J., and Lippard, S.J. 2008. Organelle‐specific zinc detection using zinpyr‐labeled fusion proteins in live cells. J. Am. Chem. Soc. 130:15776–15777.
  Uttamapinant, C., White, K.A., Baruah, H., Thompson, S., Fernandez‐Suarez, M., Puthenveetil, S., and Ting, A.Y. 2010. A fluorophore ligase for site‐specific protein labeling inside living cells. Proc. Natl. Acad. Sci. U.S.A. 107:10914–10919.
  Wombacher, R. and Cornish, V.W. 2011. Chemical tags: applications in live cell fluorescence imaging. J. Biophotonics 4:391–402.
  Zhang, C.J., Li, L., Chen, G.Y., Xu, Q.H., and Yao, S.Q. 2011. One‐ and two‐photon live cell imaging using a mutant SNAP‐Tag protein and its FRET substrate pairs. Org. Lett. 13:4160–4163.
Internet Resources
  http://www.neb.com
  For an extensive description of the SNAP‐tag labeling system, building blocks for custom probe synthesis, vectors, and probes, see the New England Biolabs Web site.
  http://www.licor.com
  For information on infrared scanning using the Odyssey system as well as reagents for custom infrared probes, see the LI‐COR Web site.
  http://www.thermoscientific.com
  For a number of NHS esters and cross‐linking agents for custom probe synthesis, see the Thermo Scientific Web site.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library