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  • Cellular Labeling (S9110)

    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 and the guidelines from your expression plasmid provider. 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 by vortexing for 10 minutes until all the SNAP-tag substrate is dissolved. Store this stock solution 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 biotin substrate. Mix substrate with medium thoroughly by pipetting up and down 10 times (necessary for reducing 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.
      Number of wells in plate Recommended Volume for Cell Labeling
      6 1 ml
      12 500 µl
      24 250 µl
      48 100 µl
      96 50 µl
      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.
    3. Wash the cells three times with tissue culture medium with serum and incubate in fresh medium for 30 minutes. Replace the medium one more time to remove unreacted SNAP-tag substrate that has diffused out of the cells.

      We recommend routinely labeling one well of non-transfected or mock-transfected cells as a negative control.

      Microscopy
      Fixation: After labeling the SNAP-tag fusion proteins with SNAP-Biotin, the cells can be fixed with 3.3% para-formaldehyde which does not result in the loss of signal. Avoid fixation using ethanol as this may lead to a high background staining of endogenous biotinylated proteins found preferentially in mitochondria.

      Detection: To visualize the SNAP-tag fusion protein in situ, permeabilize the cells with 0.5% Triton in PBS and block the cells with 1% BSA in PBS containing 0.5% Triton. Incubate the fixed cells with an appropriate streptavidin/avidin conjugate (e.g. streptavidin-fluorophore) and image the cells according to the instructions supplied with the conjugate.

      Western Blotting
      Biotinylated proteins from cell lysates can be visualized on Western Blots using standard streptavidin-based detection reagents. For Western blotting experiments, it may be more efficient to label the SNAP-tag fusion proteins after lysis of the cells in the lysate. One may also use the Anti-SNAP-tag Antibody (NEB #P9310).

      Optimizing Labeling
      Optimal substrate concentrations and reaction times range from 1–10 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 1 and 5 µM substrate and 30 minutes reaction time. 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 in live cells 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 processing the cells for imaging or blotting immediately after the labeling reaction.

      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 prior to the first wash step (Step 3) as a DNA counterstain for nuclear visualization and leave this on the cells for 2 minutes prior to completing the wash steps. Counterstaining of cells is also possible after fixation and permeabilization.

      Immunocytochemistry
      Antibody labeling of the fusion protein 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.

      Troubleshooting for Cellular Labeling
      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, Anti-SNAP-tag Antibody (NEB #P9310) can be used. 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. Improving the protein expression may also improve the signal. If the protein has limited stability in the cell, it may help to analyze the samples immediately after labeling.