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.

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

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.

Videotutorial

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

SNAP-tag:

Multiplex tagging Tools for the Study of Protein Dynamics and beyond

Download Presentation (PDF)

A complete list of all SNAP products including all available Fluorophores can be found in our brochure „Cellular Imaging and Analysis“

Download brochure (PDF)

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
PRODUCT Prod.Nr.: SIZE INFO
SNAP-Cell 430™ S9109S 50 nmol NEB Shop_icon NEB Info_icon
SNAP-Cell 505-Star S9103S 50 nmol NEB Shop_icon NEB Info_icon
SNAP-Cell Block™ S9106S 100 nmol NEB Shop_icon NEB Info_icon
SNAP-Cell Oregon Green™ S9104S 50 nmol NEB Shop_icon NEB Info_icon
SNAP-Cell TMR-Star™ S9105S 30 nmol NEB Shop_icon NEB Info_icon
SNAP-Cell® 647SiR S9102S 30 nmol NEB Shop_icon NEB Info_icon
SNAP-Surface 488™ S9124S 50 nmol NEB Shop_icon NEB Info_icon
SNAP-Surface 549™ S9112S 50 nmol NEB Shop_icon NEB Info_icon
SNAP-Surface 594 S9134S 50 nmol NEB Shop_icon NEB Info_icon
SNAP-Surface Alexa Fluor® 488 S9129S 50 nmol NEB Shop_icon NEB Info_icon
SNAP-Surface Alexa Fluor® 546™ S9132S 50 nmol NEB Shop_icon NEB Info_icon
SNAP-Surface Alexa Fluor® 647™ S9136S 50 nmol NEB Shop_icon NEB Info_icon
SNAP-Surface Block™ S9143S 200 nmol NEB Shop_icon NEB Info_icon
SNAP-Surface® 649 S9159S 50 nmol NEB Shop_icon NEB Info_icon
SNAP-tag™ Purified Protein P9312S 50 µg NEB Shop_icon NEB Info_icon
SNAP-Capture Magnetic Beads™ S9145S 2 ml NEB Shop_icon NEB Info_icon
SNAP-Capture Pull-Down Resin™ S9144S 2 ml NEB Shop_icon NEB Info_icon
SNAP-Biotin™ S9110S 50 nmol NEB Shop_icon NEB Info_icon
Anti-SNAP-tag Antibody (Polyclonal) P9310S 100 µl NEB Shop_icon NEB Info_icon
CLIP-Biotin™ S9221S 50 nmol NEB Shop_icon NEB Info_icon
CLIP-Cell 505™ S9217S 50 nmol NEB Shop_icon NEB Info_icon
CLIP-Cell TMR-Star™ S9219S 30 nmol NEB Shop_icon NEB Info_icon
CLIP-Surface 488™ S9232S 50 nmol NEB Shop_icon NEB Info_icon
CLIP-Surface 547™ S9233S 50 nmol NEB Shop_icon NEB Info_icon
CLIP-Surface 647™ S9234S 50 nmol NEB Shop_icon NEB Info_icon
BG-GLA-NHS S9151S 2 mg NEB Shop_icon NEB Info_icon
BG-Maleimide S9153S 2 mg NEB Shop_icon NEB Info_icon
BG-PEG-NH2 S9150S 2 mg NEB Shop_icon NEB Info_icon
pCLIPf Vector N9215S 20 µg NEB Shop_icon NEB Info_icon
pSNAP-tag(T7)-2 Vector N9181S 20 µg NEB Shop_icon NEB Info_icon
pSNAPf Vector N9183S 20 µg NEB Shop_icon NEB Info_icon

As of: 01.01.2024

Publications (click to expand)

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.

Troubleshooting

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)

Further information can be found in our Technical Resources section or at neb.com. Information on trademarks can be found here.