Labeling in Solution (S9234)
Overview
Dissolve the vial of CLIP-Surface 647 (50 nmol) in 17 μl of DMSO to yield a stock solution of 3 mM CLIP-tag substrate. Mix for 10 minutes until all the CLIP-tag substrate is dissolved.
Protocol
- 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.
- Add 3 mM CLIP-tag substrate solution to a total volume of 1% of the volume of the protein solution. 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 unreacted 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.
Notes for Labeling in Solution
We recommend the routine addition of 1 mM DTT to all buffers for 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.
CLIP-tag fusion proteins can be purified before labeling, but the labeling reaction also works in non-purified protein solutions (including cell lysates).
Troubleshooting for Labeling in Solution
Labeling Reaction
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).
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 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.
