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.
Dissolve one vial of CLIP-tag substrate (30 nmol) in 50 µl DMSO to yield a labeling stock solution of 0.6 mM CLIP-tag substrate in DMSO. Mix by vortexing for 10 minutes until all the CLIP-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 the requirements. The substrate is soluble in DMSO up to at least 10 mM.
- Dilute the labeling stock solution 1:200 in medium to yield a labeling medium of 3 µM dye substrate. Mix dye with medium thoroughly by pipetting up and down 10 times (necessary for reducing backgrounds). For best performance, add the CLIP-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 CLIP-tag substrate than will be consumed within one hour.
- 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.
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
- Wash the cells three times with tissue culture medium with serum and incubate them in fresh medium for 30 minutes. Replace the medium one more time to remove unreacted CLIP-tag substrate that has diffused out of the cells.
- Image the cells using an appropriate filter set. CLIP-tag fusion proteins labeled with CLIP-Cell TMR-Star should have an excitation maximum at 554 nm and an emission maximum at 580 nm, and can be imaged with standard rhodamine filter sets.
We recommend routinely labeling one well of non-transfected or mock-transfected cells as a negative control.
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-tag substrate, bromothenylcytosine (CLIP-Cell Block). 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).
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
The turnover rates of the CLIP-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 CLIP-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 CLIP-tag label with any fixation method.
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 for nuclear visualization. Counterstaining of cells is also possible after fixation and permeabilization.
Antibody labeling can be performed after CLIP-tag labeling and fixation of the cells according to standard protocols without loss of the CLIP-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
If no labeling is seen, the most likely explanation is that the fusion protein is not expressed. Verify the 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 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, following the guidelines 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.
Background fluorescence may be controlled by reducing the concentration of CLIP-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 CLIP-tag from the N- to the C-terminus or vice versa.
Photobleaching is not generally a problem as the CLIP-Cell TMR-Star substrate is very photostable. However, if problems with photobleaching are experienced, addition of a commercially available anti-fade reagent may be helpful.