ACP-tag and MCP-tag fusion proteins are expressed by transient transfection. For expression of fusion proteins with the ACP-tag or MCP-tag refer to instructions supplied with the pACP-tag(m)-2 and pMCP-tag(m) plasmids. For cell culture and transfection methods, refer to established protocols.
Dissolve one vial of CoA substrate (50 nmol) in 50 µl of DMSO to give a labeling stock solution of 1 mM CoA substrate. Mix by vortexing for 10 minutes until all the 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 substrate is soluble up to at least 10 mM.
- Dilute the substrate stock solution 1:200 in medium to a final concentration of 5 µM. Mix substrate with medium thoroughly by pipetting up and down 10 times. For best performance, add the CoA substrate to complete medium, including serum (0.5% BSA can be used for experiments carried out in serum-free media). Add MgCl2 (1:100) to a final concentration of 10 mM. Finally, add ACP Synthase or SFP Synthase to a final concentration of 1 μM, a dilution of 1:40. Do not prepare more medium with substrate, MgCl2, and synthase than you will consume within one hour.
- Replace the medium on the cells expressing a ACP-tag or MCP-tag fusion protein with the labeling medium and incubate at 37°C, 5% CO2 for 60 minutes.
These recommendations are for culturing cells in polystyrene plates. For confocal imaging, we recommend using chambered coverglass such as Lab-Tek II Chambered Coverglass which is available in a 1, 2, 4 or 8 well format.
|Number of wells in plate
||Recommended Volume for Cell Labeling
- Wash the cells three times with tissue culture medium with serum.
- We recommend routinely labeling one well of nontransfected or mock transfecred cells as a negative control.
After labeling the ACP-tag or MCP-tag fusion proteins with CoA-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 ACP-tag or MCP-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 ACP-tag or MCP-tag fusion proteins after lysis of the cells.
Optimal substrate concentrations and reaction times range from 1–10 µM and 30 minutes to overnight, respectively, depending on experimental conditions and expression levels of the ACP-tag or MCP-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 ACP-tag or MCP-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 CoA substrate for simultaneous microscopic detection. We routinely add 5 μM Hoechst 33342 (nuclear stain) for 2 minutes 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 ACP-tag or MCP-tag labeling and fixation of the cells according to standard protocols without loss of the 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.
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 ACP-tag or MCP-tag fusion protein. An ACP-tag or MCP-tag control plasmid may also be used as a positive control.
Weak labeling may be caused by insufficient exposure of the fusion protein to the substrate. Try increasing the concentration of CoA Biotin 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.