- Dissolve the vial of CoA substrate (50 nmol) in 50 μl of fresh DMSO to yield a labeling stock solution of 1 mM CoA substrate. Mix by vortexing for 10 minutes until all the CoA substrate is dissolved. Dilute this 1 mM stock solution 1:4 in fresh DMSO to yield a 250 μM stock for labeling proteins in vitro.
- Set up the reactions, in order, as follows:
|1 M HEPES
|50 mM DTT
|50 mM MgCl2
|40 µM ACP
|50 µM ACP-tag Purified Protein
|250 µM CoA
- Incubate in the dark for 60 minutes at 37°C.
- Run sample on the SDS-PAGE gel and detect using a fluorescent gel scanner or store samples at -20°C or -80°C in the dark.
Removal of Unreacted Substrate (Optional)
After the labeling reaction, the unreacted substrate can be separated from the labeled CoA fusion protein by gel filtration or dialysis. Please refer to the vendor’s instructions for the separation tools used.
Notes for Labeling in vitro
We recommend the routine addition of 1 mM DTT to all buffers used for handling, labeling and storage of the ACP- or MCP-tag. The stability of the ACP- or MCP-tag is improved in the presence of reducing agents; however it can also be labeled in their absence, if handling at temperatures above 4°C is minimized.
ACP- or MCP-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 vitro
If solubility problems occur with the ACP- or MCP-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 the 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 ACP-tag/MCP-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: Increase 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. Both approaches may be combined.
If the ACP- or MCP-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 ACP- or MCP-tag fusion protein, and store the fusion protein at –20°C.
Using less than the recommended amount of substrate stock solution can significantly slow down the reaction rate.
Loss of Activity of Protein of Interest
If the fusion protein is particularly sensitive to degradation or to loss of activity, try reducing the labeling time or decreasing the labeling temperature. We recommend overnight incubation when labeling at 4°C.