Protocol for Labeling ACP- or MCP-tag Fusion Proteins with COA-SH Substrates (S9352)
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.
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
Protocol for Labeling
- 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.
routinely labeling one well of non-transfected or mock-transfected cells as a
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.
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
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
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
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
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
Signal strongly reduced after short time
the fluorescence signal decreases rapidly, it may be due to instability of the
fusion protein. The signal may be stabilized by fixing the
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.