Protocol for Labeling ACP- or MCP-tag Fusion Proteins with CoA Substrates.
ACP-tag or MCP-tag fusion proteins can be expressed by transient transfection. For expression of fusion proteins with the ACP-tag or MCP-tag refer to the guidelines with the expression plasmid. For cell culture and transfection methods, refer to established protocols.
Dissolve one vial of CoA substrate (50 nmol) in 50 µl of DMSO to yield a solution of 1 mM CoA substrate in DMSO. Mix for 10 minutes until all the CoA 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.
Thaw one vial or aliquot (see below) of ACP Synthase or SFP Synthase and keep on ice. If you will not use all the ACP Synthase or SFP Synthase immediately (makes 5 ml of labeling medium), aliquot the remaining concentrated ACP Synthase or SFP Synthase into single use aliquots and freeze these aliquots at 80°C. It is essential to avoid freeze-thaw cycles with the ACP Synthase or SFP Synthase. Continue immediately with Step 1 below.
Protocol for Labeling Reaction
- Dilute the CoA substrate stock solution 1:200 in medium resulting in a labeling medium of 5 μM. For optimal results, add CoA substrate to complete medium, including serum. Add MgCl2 to a final concentration of 10mM. 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 ACP-Substrate, MgCl2, and ACP Synthase or SFP Synthase than you will consume within one hour.
- Replace the medium on the cells expressing an ACP-tag or MCP-tag fusion protein with the labeling medium and incubate at 37°C, 5% CO2 for 30 minutes.
- Wash the cells three times with tissue
culture medium with serum.
- Image the cells using an appropriate
filter set. For example, ACP-tag or MCP-tag fusion proteins labeled with CoA-488
should have an excitation maximum at 502 nm and an emission maximum at 522 nm,
and can be imaged with standard fluorescein filter sets.
We recommend routinely labeling one well of non-transfected or mock-transfected cells as a negative control.
The substrate concentration can be varied between 2 and 20 μM depending on the experimental conditions, expression levels of the ACP-tag or MCP-tag fusion protein, and incubation time with the substrate. Best results are usually obtained at concentrations between 5 and 10 µM. An increase of the substrate concentration usually results in a higher background and does not necessarily increase the signal to background ratio.
The incubation time can be varied between 10 and 60 minutes depending on the experimental condi-tions, expression levels of the ACP-tag fusion protein and substrate concentration. We recommend routine incubation times of 30 minutes. Longer incubation times tend to result in stronger background staining and do not necessarily increase the signal to background ratio.
Stability of labeling
The turnover rates of the ACP-tag or MCP-tag fusion protein under investigation may vary widely depending on the fusion partner. Where protein turnover is rapid, we recommend analyzing the cells under the microscope immediately after the labeling reaction or, if possible, fixing the cells directly after labeling.
Fixation of cells
The literature shows that after labeling the ACP-tag or MCP-tag fusion proteins, cells can be fixed with para-formaldehyde without loss of signal (1). We are not aware of any incompatibility of the ACP-tag labels with other fixation methods.
Cells can be counterstained with any live-cell dye that is compatible with the fluorescent properties of the CoA substrate for simultaneous microscopic detection. We routinely add 5 µM Hoechst 33342 to the labeling medium as a DNA counterstain.
The literature shows that antibody labeling at the surface of living cells after ACP-tag or MCP-tag labeling is possible (1). Antibody labeling after fixation of the cells should also be possible according to standard protocols without loss of the ACP-tag or MCP-tag signal (see fixation of cells). 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.
Experimental conditions that do not allow fetal calf serum
If fetal calf serum has to be omitted due to the experimental setup, the labeling can be done in medium without serum. Higher background levels might be observed because fetal calf serum in the labeling solution reduces the background staining. We recommend reevaluating the dye concentration and incubation time if this is a problem. The addition of 0.5% BSA may be helpful in some cases to block non-specific background.
If no labeling is seen, there is probably a problem with the expression of your fusion protein. Verify your transfection method to confirm that the cells contain the fusion gene of interest. If this is con-firmed, check for expression of the ACP-tag or MCP-tag fusion protein.
Weak labeling may be caused by insufficient exposure of the fusion protein to the substrate. Try increasing the concentration of CoA substrate, ACP Synthase, or SFP Synthase, 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.
Background fluorescence may be controlled by reducing the concentration of CoA substrate used, and by shortening the incubation time. The presence of fetal calf serum or BSA during the labeling incubation should reduce non-specific binding of substrate to surfaces. Addition of DNAse I (10 µM/ml final concentration) may also help reducing the background that may be caused by non-transfected plasmid DNA aggregating at the surface of cells.
Signal strongly reduced after short time
If the fluorescence signal decreases rapidly, it may be due to instability of the fusion protein. The signal may be stabilized by fixing the cells.
Photobleaching is not generally a problem as the CoA-Substrate is very photostable. However, if you experience problems with photobleaching, addition of a commercially available anti-fade reagent may be helpful.