Preparation of Labeling Stock Solution
Dissolve one vial of SNAP-tag substrate (10 nmol) in 10 μl of DMSO to yield a labeling stock solution of 1 mM. Mix for 10 minutes until all the SNAP-tag substrate is dissolved. Store this stock solution in the dark at 4°C, or for extended storage at -20°C. Different stock concentrations can be made, depending on your requirements. The substrates are soluble up to at least 10 mM.
- Protocol for Cell Surface Labeling Reaction
Dilute the labeling stock solution 1:200 in medium to yield a labeling medium of 5 μM SNAP-Surface 488 or SNAP-Surface 549. Mix dye with medium thoroughly by pipetting up and down 10 times (necessary for clean backgrounds). For best performance, add the SNAP-tag substrate to complete medium, including serum (0.5% BSA can be used for experiments carried out in serum-free media). Do not prepare more medium with SNAP-tag substrate than you will consume within one hour.
These recommendations are for culturing cells in polystyrene plates. For confocal imaging, we recommend using chambered coverglass such as Lab-Tek II Chambered Coverglass which is available in a 1, 2, 4 or 8 well format from Nunc.
|Number of wells in plate
||Recommended Volume for Cell Labeling
- Replace the medium on the cells expressing a SNAP-tag fusion protein with the SNAP-tag labeling medium and incubate at 37°C, 5% CO2 for 30 minutes.
- Wash the cells three times with tissue culture medium containing serum.
- Image the cells using an appropriate filter set. SNAP-tag fusion proteins labeled with SNAP-Surface 488 should have an excitation maximum at 506 nm and an emission maximum at 526 nm, and can be imaged with standard fluorescein filter sets. SNAP-tag fusion proteins labeled with SNAP-Surface 549 should have an excitation maximum at 553 nm and an emission maximum at 566 nm, and can be imaged with standard TAMRA or Cy3 filter sets.
- We recommend routinely labeling one well of non-transfected or mocktransfected cells as a negative control.
Notes for Cellular Labeling
Blocking Unreacted SNAP-tag with SNAP-Surface Block
In many cases the labeling of a non-transfected cell sample or a mocktransfected cell sample will be completely sufficient as a control. In some cases, however, it may be desirable to block the SNAP-tag activity in a cell sample expressing the SNAP-tag fusion protein to generate a control. This can be achieved using the included nonfluorescent non-cell-permeable SNAP-tag substrate, SNAP-Surface Block (C8-propanoic acid benzylguanine, CBG). SNAP-Surface Block may also be used in pulse-chase experiments to block the SNAP-tag reactivity during the chase between two pulse-labeling steps.
Optimal substrate concentrations and reaction times range from 2–20 μM and 15–60 minutes, respectively, depending on experimental conditions and expression levels of the SNAP-tag fusion protein. Best results are usually obtained at concentrations between 2 and 5 μM substrate and 30 minutes reaction time. Increasing substrate concentration and reaction time usually results in a higher background without necessarily increasing the signal to background ratio.
Stability of Signal
The turnover rates of the SNAP-tag fusion protein under investigation may vary widely depending on the fusion partner. We have seen half-life values ranging from less than one hour to more than 12 hours. Where protein turnover is rapid, we recommend analyzing the cells under the microscope immediately after the labeling reaction or, if the application allows it, fixing the cells directly after labeling.
Fixation of Cells
After labeling the SNAP-tag fusion proteins, the cells can be fixed with standard fixation methods such as para-formaldehyde, ethanol, methanol, methanol/acetone etc., without loss of signal. We are not aware of any incompatibility of the SNAP-tag label with any fixation method.
Cells can be counterstained with any live-cell dye that is compatible with the fluorescent properties of the SNAP-tag substrate for simultaneous microscopic detection. We routinely add 5 μM Hoechst 33342 to the labeling medium during the 30 minute incubation (Step 2 above) as a DNA counterstain for nuclear visualization. Counterstaining of cells is also possible after fixation and permeabilization.
Antibody labeling can be performed after SNAP-tag labeling and fixation of the cells according to standard protocols without loss of the SNAP-tag signal. The fixation conditions should be selected based on experience with the protein of interest. For example some fixation methods destroy epitopes of certain proteins and therefore do not allow antibody staining afterwards.
Problems with Cellular Labeling
If no labeling is seen, the most likely explanation is that the fusion protein is not expressed. Verify your transfection method to confirm that the cells contain the fusion gene of interest. If this is confirmed, check for expression of the SNAP-tag fusion protein. If no antibody against the fusion partner is available, a commercial anti-AGT antibody can be used (Chemicon, mouse Anti-MGMT [O6- methylguanine-DNA methyltransferase] Monoclonal Antibody, Clone MT3.1, Catalog number MAB16200). Alternatively, SNAP-Vista Green (NEB #S9147) can be used to confirm presence of SNAP-tag fusion in cell extracts following SDS-PAGE, without the need for Western blotting. An alternate explanation is that the SNAP-tag is fused to the end of the protein that is not localized on the extracellular surface of the cell membrane; reversing the orientation of the fusion protein may solve this problem.
Weak labeling may be caused by insufficient exposure of the fusion protein to the substrate. Try increasing the concentration of SNAP-tag substrate and/ or the incubation time within the range of 2–20 μM and 15–60 minutes, respectively. Alternatively the protein may be poorly expressed and/or turn over rapidly. If the protein has limited stability in the cell, it may help to analyze the samples immediately after labeling.
Background fluorescence may be controlled by reducing the concentration of SNAP-tag substrate used, and by shortening the incubation time. The presence of fetal calf serum or BSA during the labeling incubation should reduce non-specific binding of substrate to surfaces.
Signal Strongly Reduced after Short Time
If the fluorescence signal decreases rapidly, it may be due to instability of the fusion protein. The signal may be stabilized by fixing the cells. Alternatively try switching the SNAP-tag from the N- to the C-terminus or vice versa. Photobleaching is generally not a problem as both SNAP-Surface 488 and SNAPSurface 549 are very photostable. However, if you experience problems with photobleaching, addition of a commercially available anti-fade reagent may be helpful.