Site‐Specific Protein Labeling with SNAP‐Tags

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

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 O ⁶ ?alkylguanine?DNA?alkyltransferase, and undergoes a self?labeling reaction to form a covalent bond with O ⁶ ?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

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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

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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 CaCl₂ and MgCl₂ ; e.g., Invitrogen) or Dulbecco's phosphate‐buffered saline (DPBS, with CaCl₂ and MgCl₂ ; 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‐NH₂ (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‐NH₂ (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‐NH₂ (Toronto Research Chemicals, http://www.trc‐canada.com/)
Dulbecco's phosphate‐buffered saline (DPBS; with CaCl₂ and MgCl₂ ; 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‐NH₂
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‐NH₂ (Toronto Research Chemicals, http://www.trc‐canada.com/)
EZ‐Link NHS‐PEG₁₂ ‐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‐NH₂
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‐NH₂ (O ⁶ ‐[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 )

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Figures

Figure 30.1.1 A protein of interest (POI) is fused to the SNAP‐tag for expression in cells or in vitro. The reaction of the SNAP‐tag with O ⁶ ‐benzylguanine (BG) derivatives results in the covalent attachment of the label to the active‐site cysteine.

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Figure 30.1.2 Diagram of the releasable SNAP‐tag probe, BG‐S‐S‐Alexa 488 (see Cole and Donaldson, ). After binding to SNAP‐tag fusion proteins on the surface of cells, the fluorophore (e.g., Alexa Fluor 488) can be released from the protein with the cell‐impermeable reducing agent tris (2‐carboxyethyl) phosphine (TCEP).

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Figure 30.1.3 An example of a pulse‐chase turnover experiment using a SNAP‐CD147 fusion construct. CD147 is a transmembrane cargo molecule that is internalized into cells via clathrin‐independent endocytosis (Eyster et al., ). Cells are labeled with the cell‐impermeable probe BG‐800, and then chased for the indicated times. Cells are lysed, and aliquots of the lysate run on SDS‐PAGE and imaged by in‐gel fluorescence in the 800‐nm channel (Odyssey, LI‐COR, http://www.licor.com). The turnover of SNAP‐CD147 over time can be seen in (A ). In (B ), the gel was fixed and stained by Coomassie blue R250. It was then imaged by infrared scanning in the 700‐nm channel. Quantitation of image intensity from both channels can be used to normalize for protein loading to obtain an accurate reflection of protein turnover. Each time point was done in triplicate. This figure was kindly provided by Darya Karabasheva (Laboratory of Cell Biology/NHLBI/NIH).

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Videos

Literature Cited

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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.

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Site‐Specific Protein Labeling with SNAP‐Tags

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited