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Description:
SNAP-Surface™ 682 is a photostable fluorescent substrate that can be used to label SNAP-Tag^® fusion proteins in solution. This substrate (BG-682) is based on the Dyomics dye DY-682 and is suitable for excitation with a 680 nm diode laser. It has an excitation maximum at 693 nm and emission maxima at 708 nm. This package includes 50 nmol of BG-632 substrate, sufficient to make 10 ml of a 5 µM SNAP-tag fusion protein labeling solution.

The SNAP-tag is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a
polypeptide based on mammalian O⁶-alkylguanine-DNA-alkyltransferase (AGT). SNAP-tag substrates are derivates of benzyl purines and benzyl pyrimidines. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.

There are two steps to using this system: sub-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. Expression of SNAP-tag fusion proteins is described in the documentation supplied with SNAP-tag plasmids. The labeling of the fusion proteins with the SNAP-tag substrate is described below.

Materials required but not supplied:
Unlabeled SNAP-tag fusion protein
Buffer for protein dilution and labeling
Device for separation of BG-682 labeled
protein from non-reacted SNAP-tag substrate, e.g. spin column
UV-Vis spectrophotometer for determination of degree of labeling
DMSO

Figure 1. Structure of BG-682 (MW 1105.22)

Figure 2. Excitation (dotted line) and emission spectra of BG-682 coupled to SNAP-tag in buffer at pH 7.5

Reaction & Storage Conditions



Storage Temperature:

• -20°C

Notes


General notes:

   1. Storage: SNAP-Surface 682 should be stored at -20°C (long term) or at 4°C in the dark (short term, less than 4 weeks). Protect the substrate from light and moisture. With proper storage at -20°C the substrate should be stable for at least two years dry or 3 months dissolved in DMSO.

Quality Control for Current Lot


Quality control values for a specific lot can be found on the datacard which accompanies each product.

Purity and characterization::
Purity of SNAP-Surface 682 was determined to be 91% by HPLC analysis. Molecular weight [M]- was determined by MS to be 1059.3 (1059.3 expected).

In vitro protein labeling::
Reaction of SNAP-Surface 682 (10 µM) with purified SNAP-tag protein (5 µM) in vitro, followed by mass spec analysis, indicated an efficiency of labeling of 90%.

Cellular protein labeling::
Cells transfected with SNAP-tag vectors expressing ADRß2 (cell surface) were labeled with 5 µM SNAP-Surface 682 for 30 minutes, and visualized by confocal microscopy. Only surface target was efficiently labeled.

Legal


Licenses/Patents/Disclaimers:
Notice to Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH AND DEVELOPMENT PURPOSES ONLY. Commercial use of this system or any components thereof requires a license from New England Biolabs, Inc., 240 County Road Ipswich, MA 01938. For detailed information, see www.neb.com/cia/legal/.

Protocol for SNAP-Surface™ 682

- Instructions for Cellular Labeling (S9139)

Overview


SNAP-tag fusion proteins can be expressed by transient or by stable transfection. For expression of fusion proteins with the SNAP-tag refer to instructions supplied with the SNAP-tag plasmids. For cell culture and transfection methods, refer to established protocols.

Dissolve one vial of SNAP-tag substrate (50 nmol) in 50 µl of DMSO to yield a labeling stock solution of 1 mM SNAP-tag substrate. 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 substrate is soluble up to at least 10 mM.

Protocol


   1. Dilute the labeling stock solution 1:200 in medium to yield a labeling medium of 5 µM dye substrate. 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.
   2. 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.

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 .
Wash the cells three times with tissue culture medium with serum.
Image the cells using an appropriate filter set. SNAP-tag fusion proteins labeled with SNAP-Surface 682 should have an excitation maximum at 693 nm and an emission maximum at 708 nm, and can be imaged with standard fluorescein filter sets.
We recommend routinely labeling one well of non-transfected or mock-transfected cells as a negative control.

Notes

Blocking Unreacted SNAP-tag with SNAP-Surface™ Block
In many cases the labeling of a non-transfected cell sample or a mock-transfected 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 a nonfluorescent cell-impermeable 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. A protocol for blocking is included with SNAP-Surface Block (NEB #S9143).

Optimizing Labeling
Optimal substrate concentrations and reaction times range from 5–10 µM and 5–15 minutes, respectively, depending on experimental conditions and expression levels of the SNAP-tag fusion protein. Increasing substrate concentration and reaction time usually results in a higher background and does not necessarily increase 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, ethanol/acetone etc., without loss of signal. We are not aware of any incompatibility of the SNAP-tag label with any fixation method.

Counterstaining
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 medium that is used for the final 30 minutes incubation (Step 3) as a DNA counterstain for nuclear visualization. Counterstaining of cells is also possible after fixation and permeabilization.

Immunocytochemistry
Antibody labeling after SNAP-tag labeling and fixation of the cells should be possible 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.

Troubleshooting

No 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.

Weak Labeling
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, following the guidelines described above. 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.

High Background
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 not generally a problem as the SNAP-Surface 682 substrate is very photostable. However, if you experience problems with photobleaching, addition of a commercially available anti-fade reagent may be helpful.

- Instructions for Labeling Proteins in vitro (S9139)


Overview


Dissolve the vial of SNAP-tag substrate (50 nmol) in 17 μl of DMSO to yield a stock solution of 3 mM SNAP-tag substrate. Pipette up and down periodically for 10 minutes until all the SNAP-tag substrate is dissolved.

Protocol


   1. Prepare a protein solution containing up to 20 μM SNAP-tag fusion protein to be labeled in an appropriate buffer containing at least 1 mM DTT. We recommend labeling at least 100 µl of protein solution per experiment.
   2. Add SNAP-tag substrate solution to a total volume of 1% of the volume of the protein solution. Carefully pipette the material up and down to mix, and vortex briefly.
   3. Incubate for 1 hour at 25°C in the dark. Alternatively incubate overnight at 4°C in the dark.
      Removal of Unreacted Substrate (optional)

      After the labeling reaction the unreacted substrate can be separated from the labeled SNAP-tag fusion protein by gel filtration or dialysis. Please refer to the vendor’s instructions for the separation tools you are using.

      Notes for Labeling in Solution

      We recommend the routine addition of 1 mM DTT to all buffers for used for handling, labeling and storage of the SNAP-tag. The stability of the SNAP-tag is improved in the presence of reducing agents; however it can also be labeled in their absence.

      Troubleshooting

      Labeling Reaction
      If solubility problems occur with your SNAP-tag fusion protein, we recommend testing a range of pH (pH 5.0–pH 10.0) and ionic strengths. The salt concentration may also need to be optimized for your particular fusion protein (50–250 mM).

      If stickiness of the fusion protein is a problem we recommend adding Tween 20 at a final concentration of 0.05% to 0.1%. The SNAP-tag activity is not affected by this concentration of Tween 20.

      If exhaustive labeling of a protein sample is not achieved using the recommended conditions, try the following protocol modifications: Double the incubation time to two hours total at 25°C or to 24 hours at 4°C; or halve the volume of protein solution labeled (50 µl of a solution containing up to 20 µM SNAP-tag fusion protein). Both approaches may be combined. If you still have poor labeling results, we recommend checking the activity of the SNAP-tag using SNAP-Vista.

      If your fusion protein is particularly sensitive to degradation or to loss of activity, you can try reducing the labeling time or decreasing the labeling temperature. If you label at 4°C we recommend over night incubation.