Labeling of Proteins in Solution (E9200)
The supplied dye substrates can also be used to label CLIP-tag fusion proteins in solution for biochemical applications including FRET, labeling of proteins used for detection, etc.
Preparation of Labeling Stock Solution
Dissolve one vial of CLIP-tag substrate in 3.3 μl of DMSO to yield a labeling stock solution of 3 mM CLIP-Cell 505 or 1.8 mM CLIP-Cell TMR-Star. Mix periodically 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 your requirements. The substrates are soluble up to at least 10 mM.
- Protocol for Solution Labeling Reaction
Prepare a protein solution containing up to 20 μM CLIP-tag fusion protein to be labeled in an appropriate buffer containing at least 1 mM DTT. The CLIP-tag labeling reaction works well between pH 5 and 10, and at NaCl concentrations from 15 mM to 1 M.
- Add CLIP-tag substrate solution to a total volume of 1% of the volume of the protein solution (final concentration 30 μM CLIP-Cell 505, or 18 μM CLIP-Cell TMR-Star). Carefully pipette the material up and down to mix, and vortex briefly.
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 you may wish to separate the nonreacted substrate from the labeled CLIP-tag fusion protein. You can use gel filtration or dialysis. Please refer to the vendor’s instructions for the separation tools you are using.
Note for Labeling in Solution: We recommend the routine addition of 1 mM DTT to all buffers used for handling, labeling and storage of the CLIP-tag. The stability of the CLIP-tag is improved in the presence of reducing agents; however it can also be labeled in their absence (e.g. for a redox-sensitive protein) if handling at temperatures above 4˚C is minimized. CLIP-tag fusion proteins can be purified before labeling, but the labeling reaction also works in non-purified protein solutions (including cell lysates).
If solubility problems occur with your CLIP-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).
Loss of Protein due to Aggregation or Sticking to Tube
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 CLIP-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 CLIP-tag fusion protein). Both approaches may be combined. If you still have poor labeling results, we recommend checking the activity of the CLIP-tag using CLIP-Vista Green (NEB #S9235).
If the CLIP-tag fusion has been stored in the absence of DTT or other reducing agent, or has been stored at 4˚C for a prolonged period, its activity may be compromised. Include 1 mM DTT in all solutions of the CLIP-tag fusion protein, and store the fusion protein at -20˚C.
Using less than the recommended amount of substrate stock solution (1%) can significantly slow down the reaction rate.
Loss of Activity of Protein of Interest
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 overnight incubation.