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Cellular Analysis (SNAP-Tag)

SNAP-tag Technology

SNAP- and CLIP-tag protein labeling systems enable the specific, covalent attachment of virtually any molecule to a protein of interest. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag® fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein.

SNAP-tag substrates are dyes, fluorophores, biotin, or beads conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag™ is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

The SNAP- or CLIP-tag is fused to the protein of interest. Labeling occurs through covalent attachment to the tag, releasing either a guanine or a cytosine moiety.

Workflow

Clone and express your protein of interest fused to the SNAP-tag once, then use with a variety of substrates for subsequent analysis.

Fluorescent Labeling of COS-7 Expressing SNAP-tag® Fusion Proteins for Live Cell Imaging

Applications

  • Simultaneous dual protein labeling inside live cells
  • Protein localization and translocation
  • Pulse-chase experiments
  • Receptor internalization studies
  • Selective cell surface labeling
  • Protein pull-down assays
  • Protein detection in SDS-PAGE
  • Flow cytometry
  • High throughput binding assays in microtiter plates
  • Biosensor interaction experiments
  • FRET-based binding assays
  • Single molecule labeling
  • Super-resolution microscopy

SNAP-tag: Multiplex tagging Tools for the Study of Protein Dynamics and beyond

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Nobel Prize in Chemistry 2014

The Nobel Prize in Chemistry 2014 was awarded to Stefan Hell, Goettingen, who has also used the SNAP-tag for his STED studies:

Confocal vs. STED microscopy of living U2-OS cell:
The cells overexpress a SNAP-tag fusion of Cep41, a microtubuli binding protein. SNAP-Cell® 647SiR was used to  detect the fusion protein.
Lukinavičius G et al. (2013) « A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. » Nat. Chem. 5(2): 132-9.

Comparison of SNAP-tag® / CLIP-tagTM Technologies to GFP

While SNAP/CLIP-tag technologies are complementary to GFP, there are several applications for which SNAP- and CLIP-self-labeling technologies are advantageous.

Application SNAP-tag/CLIP-tag GFP and other fluorescent proteins
Time-resolved fluorescence Fluorescence can be initiated upon addition of label Color is genetically encoded and always expressed. Also, photoactivatable fluorescent proteins require high intensity laser light, which may activate undesired cellular pathways (e.g., apoptosis)
Pulse-chase analysis Labeling of newly synthesized proteins can be turned off using available blocking reagents (e.g., SNAP-Cell® Block) Fluorescence of newly synthesized proteins cannot be quenched to investigate dynamic processes
Ability to change colors A single construct can be used with different dye substrates to label with multiple colors Requires separate cloning and expression for each color
Surface specific labeling Can specifically label subpopulation of target protein expressed on cell surface using non-cell permeable substrates Surface subpopulation cannot be specifically visualized
Visualizing fixed cells Resistant to fixation; strong labeling Labile to fixation; weak labeling
Pull-down studies “Bait” proteins can be covalently captured on BG beads Requires anti-GFP antibody to non-covalently capture “bait” protein, complicating downstream analysis
Live animal imaging Near-IR dyes are available, permitting deep tissue visualization Limited to visible wavelengths

Troubleshooting

 Application

 Problem

 Possible Cause

 Solution

  Cellular Labeling

 No labeling  Fusion protein
not expressed
  1. Verify transfection
  2. Check expression of fusion protein via Western blot or SDS-PAGE with Vista Green label
 Weak labeling  Poor expression and/or insufficient exposure of fusion protein to substrate
  1. Increase substrate concentration
  2. Increase incubation time
 Rapid turnover of fusion protein
  1. Analyze samples immediately or fix cells directly after labeling
  2. Label at lower temperature (4°C or 16°C)
 High background  Non-specific binding of substrates
  1. Reduce substrate concentration and/or incubation time
  2. Allow final wash step to proceed for up to 2 hours
  3. Include fetal calf serum or BSA during labeling
 Signal strongly reduced after short time  Instability of fusion protein
  1. Fix cells
  2. Switch tag from N-terminus to C-terminus or vice versa
 Photobleaching
  1. Add commercially available anti-fade reagent
  2. Reduce illumination time and/or intensity

Labeling in Solution

 Precipitation  Insoluble fusion
  1. Test from pH 5.0 to 10.0
  2. Optimize salt concentration [50 to 250 mM]
  3. Add 0.05 to 0.1% Tween 20
 Weak or no labeling  Exhaustive labeling has not been achieved
  1. Increase incubation time to 2 hrs at 25°C or 24 hrs at 4°C
  2. Reduce the volume of protein solution labeled
  3. Check expression of fusion protein via SDS-PAGE with Vista Green label
 Loss of activity  Instability of fusion protein
  1. Reduce labeling time
  2. Decrease labeling temperature (4°C or 16°C)

Starter Kits

Product NEB#. Plasmid Fluorophore Block Applications Price
SNAP-Cell® Starter Kit E9100S pSNAPf Vector SNAP-Cell® 505, SNAP-Cell® TMR-Star SNAP-Cell® Block – Intracellular labeling – Cell surface labeling in vitro analysis 252 €
SNAP-Surface® Starter Kit E9120S pSNAPf Vector SNAP-Surface® 488, SNAP-Surface® 549 SNAP-Surface™ Block – Cell surface labeling in vitro analysis 252 €
CLIP-Cell™ Starter Kit E9200S pCLIPf Vector CLIP-Cell™ 505, CLIP-Cell™ TMR-Star CLIP-Cell™ Block – Intracellular labeling – Cell surface labeling in vitro analysis 252 €
CLIP-Surface™ Starter Kit E9230S pCLIPf Vector CLIP-Surface™ 488, CLIP-Surface™ 547 CLIP-Cell™ Block – Cell surface labeling in vitro analysis 252 €
ACP-Surface Starter Kit E9300S pACP-tag(m)-2 Vector CoA 488, CoA 547 N/A – Cell surface labeling in vitro analysis 252 €

Publications

STED
Guzmán, C. et al. (2014) « The efficacy of Raf kinase recruitment to the GTPase H-ras depends on H-ras membrane conformer specific nanoclustering » J. Biol. Chem. 289, 9519-9533.
Stagge, F. et al. (2013) « Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells » PLoS One 8(10): e78745.
Lukinavičius, G. et al. (2013) « Selective Chemical Crosslinking Reveals a Cep57-Cep63-Cep152 Centrosomal Complex » Curr. Biol. 23, 265-270.
Lukinavičius, G. et al. (2013) « A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins » Nat. Chem. 5, 132-139.
Pellett P. A. et al. (2011) « Two-color STED microscopy in living cells. » Biomed. Opt. Expr. 2, 2364-2371.
Testa I. et al. (2010) « Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength » Biophys. J. 99, 2686-94.
Hein B. et al. (2010) « Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins. » Biophys. J. 98, 158–163.

STORM
Liu, Z. et al. (2014) « Super-resolution imaging and tracking of protein-protein interactions in sub-diffraction cellular space » Nat. Commun. 5, 4443.
Perkovic, M. et al. (2014) « Correlative Light- and Electron Microscopy with chemical tags » J. Struct. Biol. 186, 205-213.
Carlini, L. et al. (2014) « Reduced Dyes Enhance Single-Molecule Localization Density for Live Superresolution Imaging » ChemPhysChem 15, 750-755.
Sateriale, A. et al. (2013) « SNAP-Tag Technology Optimized for Use in Entamoeba histolytica » PLoS One 8(12), e83997.
Lukinavičius, G. et al. (2013) « A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins » Nat. Chem. 5, 132-139.
Malkusch, S. et al. (2013) « Single-molecule coordinate-based analysis of the morphology of HIV-1 assembly sites with near-molecular spatial resolution » Histochem. Cell Biol. 139, 173-179.
van de Linde, S. et al. (2011) « Direct stochastic optical reconstruction microscopy with standard fluorescent probes » Nat. Protoc. 6, 991-1009.
Eckhardt M. et al. (2011) « A SNAP-Tagged Derivative of HIV-1-A Versatile Tool to Study Virus-Cell Interactions. » PLoS One 6(7), e22007.
Jones S. A. et al. (2011) « Fast, three-dimensional super-resolution imaging of live cells. » Nat. Methods 8, 499-505.
Klein T. et al. (2011) « Live-cell dSTORM with SNAP-tag fusion proteins. » Nat. Methods 8, 7-9.
Dellagiacoma C. et al. (2010) « Targeted Photoswitchable Probe for Nanoscopy of Biological Structures » ChemBioChem 11, 1361–1363.

PALM
Benke, A. et al. (2012) « Multicolor Single Molecule Tracking of Stochastically Active Synthetic Dyes » Nano Lett. 12, 2619-2624.
Banala, S. et al. (2012) « A caged, localizable rhodamine for superresolution microscopy » ACS Chem. Biol. 7, 289-293

RLS-SRM
Zhao, Z. W. et al. (2014) « Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy » Proc. Natl. Acad. Sci. USA 111, 681-686.

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