Preparation of Labeling Stock Solution
Dissolve one vial of CoA substrate (10 nmol) in 10 µl of fresh DMSO to yield a labeling stock solution of 1 mM. Mix by vortexing 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 substrates are soluble up to at least 10 mM.
- Protocol for Cell Surface Labeling Reaction
Dilute the labeling stock solution 1:200 in medium to yield a labeling medium of 5 µM CoA substrate. Mix dye with medium thoroughly by pipetting up and down 10 times (necessary for reducing backgrounds). 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 to a final concentration of 10 mM (1:100 dilution of supplied 1 M stock solution). Finally, add SFP Synthase (supplied) or ACP Synthase (NEB #P9301 ) 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.
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 from Nunc.
|Number of wells in plate
||Recommended Volume for Cell Labeling
- Replace the medium on the cells expressing an ACP-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 containing serum.
- Image the cells using an appropriate filter set. ACP-tag and MCP-tag fusion proteins labeled with CoA 488 should have an excitation maximum at 506 nm and an emission maximum at 526 nm, and can be imaged with standard fluorescein filter sets. ACP-tag and MCP-tag fusion proteins labeled with CoA 547 should have an excitation maximum at 554 nm and an emission maximum at 568 nm, and can be imaged with standard TAMRA or Cy3 filter sets.
- We recommend routinely labeling one well of non-transfected or mock-transfected cells as a negative control.
Notes for Cellular Labeling
Dual labeling of ACP-tag and MCP-tag fusion proteins with different substrates
The included SFP Synthase will efficiently modify both ACP-tag and MCP-tag fusion proteins displayed on the cell surface, whereas the ACP Synthase (NEB #P9301S ) will modify ACP-tag fusions only. This permits sequential dual labeling of two different surface-localized proteins in the same cell, one protein expressed as an ACP-tag fusion and the other as an MCP-tag fusion.
Simply carry out the labeling procedure described above using ACP Synthase and one CoA substrate (to label the ACP-tag fusion), then wash 3 times with label-free medium to remove unreacted substrate and the synthase. Then repeat the labeling procedure with a second CoA substrate and the SFP Synthase to label the MCP-tag fusion.
Optimal substrate concentrations and reaction times range from 1–20 µM and 30–60 minutes, 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 1–5 µM substrate and 30 minutes reaction time. Increasing substrate concentration and reaction time usually results in a higher background without necessarily increasing the signal to background ratio.
Stability of Signal
The turnover rates of the ACP-tag or MCP-tag fusion protein 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 analyzing the cells under the microscope immediately after the labeling reaction or, if the application allows it, fixing the cells directly after labeling. As an alternative to visualize proteins with fast turnover rates, ACP-tag and MCP-tag fusion proteins can be labeled at lower temperatures (4 or 16°C). Labeling times may need to be optimized.
Fixation of Cells
After labeling the ACP-tag or MCP-tag fusion proteins, the cells can be fixed with standard fixation methods such as para-formaldehyde, ethanol, methanol, methanol/acetone etc., without loss of signal. We are not aware of any incompatibility of the ACP-tag or MCP-tag with any fixation method.
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 after the 30 minute incubation (step 2 above) as a DNA counterstain for nuclear visualization of live cells. DAPI should be used for counterstaining of cells after fixation and permeabilization.
Antibody labeling can be performed after ACP/MCP-tag labeling and fixation of the cells according to standard protocols without loss of 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.
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 carried out in medium without serum. Higher background levels might be observed because fetal calf serum in the labeling solution reduces the background staining. We recommend re-evaluating 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.
- Problems with Cellular Labeling
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 (e.g. by Western blot). If no antibody against the fusion partner is available, label cells with CoA Biotin (NEB #S9351 ) following the procedure above, then run a Western blot and probe with commercially available labeled streptavidin or anti-biotin antibody.
An alternate explanation may be that the terminus of the protein fused to the tag is not localized on the extracellular surface of the cell membrane and therefore the ACP-tag or MCP-tag is not available for labeling by ACP or SFP Synthase; placing the tag at the other terminus of the protein (keeping the secretion peptide at the N-terminus) may alleviate this problem.
Weak labeling may be caused by insufficient exposure of the fusion protein to the substrate. Try increasing the concentration of CoA substrate and/or the incubation time within the range of 1–20 µM and 30–60 minutes, respectively. Alternatively the protein may be poorly expressed and/or turn over rapidly. 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 CoA substrate to surfaces. Addition of DNAse I (10 μM/ml final concentration) may also help reduce the background that may be caused by non-transfected plasmid DNA aggregating at the surfaces 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. Alternatively try switching the ACP-tag or MCP-tag from the N- to the C-terminus or vice versa. Photobleaching is generally not a problem as both CoA 488 and CoA 547 are very photostable. However, if you experience problems with photobleaching, addition of a commercially available anti-fade reagent may be helpful.