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  • Instructions for Use with SFP Synthase (P9302)

    Overview

    ACP-tag or MCP-tag fusion proteins can be expressed by transient transfection. For expression of fusion proteins with the ACP-tag or the MCP-tag, refer to instructions provided with pACP-tag(m)-2 Vector (NEB #N9322 ) or pMCP-tag(m) Vector (NEB #N9317 ) cloning 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 solution of 1 mM CoA substrate. 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 the requirements. The substrate is soluble up to at least 10 mM.

    Protocol

    1. Dilute 1 mM CoA substrate stock solution 1:200 in culture medium (a final concentration of 5 μM). We obtain best performance by adding the substrate to complete medium, including serum. Add MgCl2 (1:100) to a final concentration of 10 mM. Finally, add the SFP Synthase to a final concentration of 1 μM, a dilution of 1:40. Do not prepare more medium with CoA substrate, MgCl2 and SFP Synthase than will be consumed within one hour. 
    2. 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 60 minutes. 

    3. Wash the cells three times with tissue culture medium with serum.

    4. Image the cells using an appropriate filter set.

    5. We recommend routinely labeling one well of non-transfected or mock-transfected cells for comparison.

      Notes for Cellular Labeling

      Optimizing Labeling
      The substrate concentration can be varied between 1 and 10 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 1 and 5 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 30 and 60 minutes depending on the experimental conditions, expression levels of the ACP-tag or MCP-tag fusion protein and substrate concentration. We recommend routine incubation times of 60 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
      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 label with any fixation method.

      Counterstaining
      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 medium prior to the first wash (Step 3) as a DNA counterstain and leave this on the cells for 2 minutes prior to completing the wash steps.

      Antibody Labeling
      Antibody labeling at the surface of living cells after ACP-tag or MCP-tag labeling is possible. Antibody labeling after fixation of the cells is also possible using 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 must be omitted due to the experimental setup, the labeling can be done in medium without serum. Higher background levels might be observed as 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.

      Troubleshooting for Cellular Labeling

      No Labeling
      If no labeling is seen, the most likely explanation is that the fusion protein is not expressed. Verify the 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 via Western blot.

      Weak Labeling
      Weak labeling may be caused by insufficient exposure of the fusion protein to the substrate. Try increasing the concentration of CoA substrate, 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.

      Weak labeling can also be caused by loss of activity of the SFP Synthase. Increasing the concentration of SFP Synthase may help.

      High Background
      Background fluorescence can 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 (5 units/ml final concentration) can help reduce the background 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. Alternatively, try switching the ACP-tag or MCP-tag from the N- to the C-terminus or vice versa.

      Photobleaching is not generally a problem as the CoA substrates are very photostable. However, if problems with photobleaching arise, addition of a commercially available anti-fade reagent may be helpful.