Cellular Labeling (S9217)


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



  2. Replace the medium on the cells expressing a CLIP-tag fusion protein with the CLIP-tag labeling medium and incubate at 37°C, 5% CO2 for 60 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


  4. Image the cells using an appropriate filter set. CLIP-tag fusion proteins labeled with CLIP-Cell 505 should have an excitation maximum at 504 nm and an emission maximum at 532 nm, and can be imaged with standard fluorescein filter sets.



    Blocking Unreacted CLIP-tag with CLIP-Cell 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 CLIP-tag activity in a cell sample expressing the CLIP-tag fusion protein to generate a control. This can be achieved using a nonfluorescent CLIP-Cell Block (bromothenylcytosine, BTC). CLIP-Cell Block may also be used in pulse-chase experiments to block the CLIP-tag reactivity during the chase between two pulse-labeling steps. A protocol for blocking is included with CLIP-Cell Block (NEB #S9220 ).

    Optimizing Labeling
    Optimal substrate concentrations and reaction times range from 1–10 µM and 30–60 minutes, respectively, depending on experimental conditions and expression levels of the CLIP-tag fusion protein. Best results are usually obtained at concentrations between 1 and 5 µM substrate and 60 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

    Fixation of Cells

    Cells can be counterstained with any live-cell dye that is compatible with the fluorescent properties of the CLIP-tag substrate for simultaneous microscopic detection. We routinely add 5 μM Hoechst 33342 to the medium prior to the final 30 minutes incubation (Step 3) as a DNA counterstain. Counterstaining of cells is also possible after fixation and permeabilization.


    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 CLIP-tag fusion protein via Western blot using Anti-SNAP-tag Antibody (NEB #P9310 ). This antibody shows high crossreactivity with the CLIP-tag and can be used for Western blot detection. Alternatively, CLIP-Vista Green (NEB #S9235 ) can be used to confirm presence of CLIP-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 CLIP-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. 

    High Background

    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 CLIP-tag from the N- to the C-terminus or vice versa. 

    Photobleaching is not generally a problem as the CLIP-Cell 505 substrate is very photostable. However, if you experience problems with photobleaching, addition of a commercially available anti-fade reagent may be helpful.