CLIP-tag fusion proteins can be expressed by transient or by stable transfection. For expression of fusion proteins with the CLIP-tag refer to instructions supplied with the CLIP-tag plasmids and the guidelines from your expression plasmid provider. For cell culture and transfection methods, refer to established protocols.
Dissolve one vial of CLIP-tag substrate (50 nmol) in 50 µl of DMSO to yield a labeling stock solution of 1 mM CLIP-tag substrate. Mix by vortexing for 10 minutes until all the CLIP-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.
- 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 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 you will consume 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.
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- 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 CLIP-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.
After labeling the CLIP-tag fusion proteins with CLIP-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.
To visualize the CLIP-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.
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 CLIP-tag fusion proteins after lysis of the cells in the lysate. One may also use the Anti-SNAP-tag Antibody (NEB #P9310).
Optimal substrate concentrations and reaction times range from 1–10 µM and 15–60 minutes to overnight, 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 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.
Cells can be counterstained with any live-cell dye that is compatible with the properties of the CLIP-tag substrate for simultaneous microscopic detection. We routinely add 5 μM Hoechst 33342 (nuclear stain) after labeling with avidin/streptavidin followed by 2 short washing steps. Counterstaining of cells is also possible with dyes that do not enter live cells after fixation and permeabilization.
Antibody labeling of the fusion protein 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 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.
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.