Labeling Protocol for SNAP-tag®

Your SNAP-tag® fusion proteins within living cells, on cell surfaces and on fixed cells can be colored with the abberior LIVE SNAP labels. These labels interact highly with the SNAP-tag® protein forming a covalent bond between label and protein.

with abberior LIVE dyes


abberior offers a variety of excellent fluorescent probes with properties optimized for the labeling of biomolecules in living specimens, for spectroscopic studies, and for, optical microscopy, particularly STED superresolution microscopy. These unique dyes & labels combine the highest brightness and photostability with easy live-cell labeling.

Our abberior LIVE SNAP labels1,2,3 can be readily used to specifically mark your SNAP-tag® fusion proteins of interest within living cells or on cell surfaces. Due to a high affinity, these labels interact with the SNAP-tag® protein and form via an enzymatic reaction a covalent bond between label and protein.


abberior SNAP labels are shipped freeze-dried in amounts of 30 nmol per vial. Upon arrival, the substances can be stored for up to one year at –20 °C. Shortly before the staining procedure dissolve the probe in DMF or DMSO. Once dissolved the stock solutions should be kept at –20 °C, protected from light and moisture.

Note: Depending on solvent quality the shelf-life of the stock solutions might be significantly reduced compared to the substances in their solid form – even if stored at –20 °C.

Staining of targets using abberior LIVE SNAP labels

The procedure described below has been successfully tested with our LIVE SNAP labels in several stably and transiently transfected adherent mammalian cultured cell lines expressing a SNAP-tag® fusion protein. This procedure has yielded consistent results in most instances but may require further optimization for particular model organisms.

Required reagents and equipment; not provided

  • DMF or DMSO
  • glass coverslips, glass-bottom dish, or similar imaging chamber with a glass thickness of ~170 µm (No. 1.5 or No. 1.5H)

    Note: We do not recommend using plastic coverslips or live-cell chambers with plastic bottoms because frequently only suboptimal imaging results are achieved. If possible, coverslips with grids, gratings, or similar should be avoided, as these structures can interfere with imaging causing aberrations that degrade image quality.
  • Live-cell imaging medium
  • Optional: Cavity slide or coverslip holder
  • Optional: silicone glue (e. g. Twinsil, Picodent)
  • Fluorescence microscope with live-cell incubator, suitable excitation light source and detection filter
  • Gene vector and transfection reagents

Staining procedure for cultured cells

The seeding time should be adjusted according to the doubling time of the particular cell line. Seed cells at the desired density in a cell culture chamber or onto coverslips before labeling. Use a standard cell culture medium that is optimal for the cell line.

For transfection of the cells, please follow the instructions of the manufacturer of the transfection reagents.

Note: Cells grown in very high densities, i.e., a confluent layer, may give rise to the high labeling background.

Staining and imaging will take place on the same day.

  1. Prewarm the live-cell imaging medium to the optimal temperature required to cultivate the desired cell line. In most cases, this is 37 °C.
  2. Please dissolve the abberior LIVE SNAP probe in DMF or DMSO for preparing a stock solution with a concentration of 1 mM.
  3. Prepare the staining solutions with final concentrations of 0.1 to 1 µM. For this, the stock solution of the abberior LIVE SNAP label is further diluted in a prewarmed cell culture medium or live-cell imaging medium.

    Note: The concentration required for proper labeling strongly depends on the specimen (tissue or cell). It can vary drastically between cell types, the expression rate of the protein tag, and experimental conditions.

    Note: Staining solutions are not stable for extended periods of time. Therefore, it is recommended to prepare enough solution for immediate use.

    Optional: The LIVE SNAP labels can be combined with our direct LIVE labels, HaloX®4, and with our STAR membrane probes. Simply add them to the staining solution for multicolor live-cell imaging.
  4. Remove the cell culture medium and rinse the cells once with a prewarmed live-cell imaging medium.
  5. Remove the live-cell imaging medium and add a sufficient amount of staining solution to the cells. Incubate for 30 min at optimal cell growth conditions (temperature, humidity, CO2-controlled environment).

    Note: Excessive staining can lead to a high background or non-specific staining.
  6. Remove the cells from the staining solution and wash the cells in a fresh imaging medium for 30 minutes.
  7. Embed the cells:
    If using cells grown on a coverslip: Simply take the coverslip out of the washing solution using tweezers. Place the coverslip in a coverslip holder and fill it with the staining solution. Alternatively, mount the coverslip (cells facing downwards) onto a slide with a cavity filled with staining solution.
    In case of imaging cells in a cavity slide, remove any excess staining solution using tissue paper. Gently press down the coverslip to prevent it from moving. The mounted sample can be sealed using silicone glue (e. g. Twinsil, Picodent).
  8. After staining and mounting, the samples should be imaged directly on a microscope equipped with a live-cell incubator.

    Note: For live-cell imaging, cells must always be kept at ambient conditions (temperature, humidity, pH, and CO2-conditions). This is particularly important for long-term measurements.


DMF N,N-Dimethylformamide
DMSO Dimethylsulfoxide
STED Stimulated Emission Depletion

1 SNAP-tag® is a registered trademark of New England Biolabs, Inc. (NEB)
2 This product is covered by one or more patents owned or controlled by New England Biolabs, Inc. (NEB), and is intended for Research Use Only (RUO)
3 abberior LIVE SNAP labels are modified by a benzylguanine substrate as a functional group.
4 HaloX® is a registered trademark of Spirochrome AG