Once the amine-containing precursor has been converted into a BC-substrate it can be used to label CLIP-tag fusion proteins. CLIP-tag fusions can be labeled in cell lysates or as purified protein. For expression of fusion proteins with the CLIP-tag, refer to instructions supplied with the CLIP-tag plasmids.
We recommend using a 1.5 fold excess of substrate over CLIP-tag fusion protein and incubating for 1 hour at room temperature. Typical concentrations are 30 µM substrate and 20 µM CLIP-tag fusion protein. The labeling incubation is followed by a separation step such as dialysis or spin column separation to remove unreacted substrate.
- Unlabeled protein samples should be stored at -20°C, or
at -80°C for long-term storage. Many proteins benefit from the addition of
glycerol for frozen storage, typically 20% v/v. Handling at temperatures above
0°C should be minimized by thawing the unlabeled protein samples shortly before
use, and keeping them on ice until just before the labeling reaction.
recommend the routine addition of 1 mM DTT to all buffers used for handling,
labeling and storage of CLIP-tag fusion proteins. The stability of the CLIP-tag
is improved in the presence of reducing agents; however it can also be labeled
in their absence.
If a particular fusion protein requires buffers
without reducing agents, pay particular attention to minimize all handling steps
of the protein above 4°C prior to the labeling reaction.
labeling reaction works well between pH 5.0 and pH 10.0. The salt concentration
may need to be optimized for your particular fusion protein, although the CLIP-tag labeling reaction has been shown to work at a broad range of ionic
strengths (NaCl concentrations between 15 mM and 1 M). If the buffer conditions
used for labeling lead to insolubility of your protein, we recommend testing a
range of pH and ionic strengths. For a range of fusion proteins we have found
ionic strengths for monovalent salts (e.g. sodium chloride) from 50 mM to 250 mM
Where stickiness of the fusion protein is a problem we
recommend adding Tween 20 at a final concen-tration of 0.05% to 0.1%. At this
concentration Tween 20 does not affect the performance of the CLIP-tag. Ionic
detergents (e.g. SDS) should be avoided.
The maximum degree of labeling
possible with the CLIP-tag is one molecule of label per molecule of fusion
- Storage and Handling of Labeled CLIP-tag Fusion
Labeled CLIP-tag fusion proteins should be stored at
concentrations of 1 mg/ml or less at 4°C protected from light. Sodium azide may
be added to 2 mM final concentration. Under these conditions the labeled protein
should be stable at 2–6°C for several months. If freezing is required, it may be
best to shock freeze by rapidly cooling the sample to a temperature
significantly below 0°C in the presence of 20% (v/v) glycerol.
protein is linked to the CLIP-tag label by a covalent bond. Therefore the
conjugates are highly stable. DTT or other reducing agent is not required in the
storage buffers. As the fusion protein is covalently labeled, labeled protein
may be detected after harsh or even denaturing treatments, such as
chromatographic or electrophoretic separation, including SDS-PAGE.
Avoid repeated freezing and thawing. Protect from light if the protein is
labeled with a fluorophore.
If exhaustive labeling of a protein sample is not achieved using the recommended conditions, try the following protocol modifications: Increase the incubation time to two hours total at 25°C or to 24 hours at 4°C; or double the ratio of label to protein in the labeling reaction. Both approaches may be combined. If you still have poor labeling results, we recommend checking the activity of the CLIP-tag.
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.
If the degree of labeling is extremely low, the labeling reaction was probably inefficient. A number of reasons can lead to such inefficient labeling:
If you added the substrate dissolved in DMSO at greater than 1% of the volume of protein solution, the higher DMSO concentration may slow down the reaction rate. At lower substrate concentrations the labeling time should be increased to 2 hours.
The activity of the CLIP-tag may have been partially or completely lost. This may be due to extended storage of non-reacted CLIP-tag fusion proteins at 4°C or greater. The sensitivity of the CLIP-tag to inactivation is also significantly increased if no reducing agent is added (such as DTT at 1 mM).
If you encounter problems with the activity we recommend thawing another sample of your protein or reexpressing and repurifying the CLIP-tag fusion protein following the advice given in the pCLIP-tag™(m) Vector (NEB #N9211) instructions.