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Description:
CoA 547 is a photostable fluorescent substrate that can be used to label ACP-tag and MCP-tag fusion proteins exposed on the surface of living cells. This cell-impermeable substrate is based on the Dyomics dye DY-547, and is suitable for standard TAMRA and Cy3 filter sets. It has an excitation maximum at 554 nm and an emission maximum at 566 nm. This package contains 50 nmol of CoA 547 substrate, sufficient to make 10 ml of a 5 μM ACP-tag or MCP-tag fusion protein labeling solution.

The ACP-tag and MCP-tag are polypeptide tags (8 kDa) based on the acyl carrier protein. MCP-tag contains two mutations (D36T and D39G). Both allow the specific, covalent attachment of virtually any molecule to a protein of interest. Substrates are derivates of coenzyme A (CoA). In the labeling reaction, the substituted phosphopantetheine group of CoA is covalently attached to a conserved serine residue of the ACP-tag or the MCP-tag by a phosphopantetheinyl transferase (SFP Synthase, or ACP Synthase).

While ACP Synthase (NEB #P9301) will preferentially modify the ACP-tag, SFP Synthase (NEB #P9302) will label both ACP-tag and MCP-tag.

Having no cysteines, the ACP-tag and the MCP-tag are particularly suited for specifically labeling cell-surface proteins, and should be useful for labeling secreted proteins with disulfide bridges such as antibodies.

There are two steps to using this system: sub-cloning and expression of the protein of interest as an ACP-tag or MCP-tag fusion, and labeling of the fusion protein using the appropriate synthase with the CoA substrate of choice. Expression of ACP- and MCP-tagged proteins is described in the documentation supplied with the pACP and pMCP plasmids, respectively. The labeling of the fusion proteins with the CoA substrate is described in the product protocol.

Materials Required but not Supplied:
ACP Synthase (NEB #P9301) for labeling ACP-tag
SFP Synthase (NEB #P9302) for labeling ACP-tag or MCP-tag
Cells expressing ACP-tag or MCP-tag fusion proteins
Tissue culture materials and media
Transfection reagents
Fluorescence microscope with suitable filter set
DMSO

Live CHO-K1 cells transiently transfected with pACP-GPI. Cells were labeled with CoA 547 (red) in the presence of ACP Synthase for 30 minutes.

Figure 1. Structure of CoA 547 (MW 1528.4)

Figure 2. Excitation (dotted line) and emission spectra of CoA 547 coupled to ACP-tag in buffer at pH 7.4.

Reaction & Storage Conditions



Storage Temperature:

• -20°C

Notes


General notes:

   1. Storage: CoA 547 should be stored at -20°C (long term) or at 4°C in the dark (short term, less than 4 weeks). Protect the substrate from light and moisture. With proper storage at -20°C the substrate should be stable for at least one year dry or 3 months dissolved in DMSO.

Usage notes:

   1. Optimizing Labeling:  Optimal substrate concentrations and reaction times range from 2–20 µM and 15–60 minutes, respectively, depending on experimental conditions and expression levels of the ACP-tag and MCP-tag fusion protein. Best results are usually obtained at concentrations between 5 and 10 µM substrate and 30 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.
   2. Stability of Labeling: The turnover and internalization rates of the ACP-tag and 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, fixing the cells directly after labeling.
   3. 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 SNAP-tag label with any fixation method.
   4. Counterstaining: Cells can be counterstained with any live-cell dye that is compatible with the fluorescent properties of the substrate for simultaneous microscopic detec-tion. We routinely add 5 µM Hoechst 33342 to the labeling medium as a DNA counterstain for nuclear visualization.
   5. Immunocytochemistry: Antibody labeling can be performed after SNAP-tag labeling and fixation of the cells according to standard protocols without loss of the SNAP-tag 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.

Quality Control for Current Lot


Quality control values for a specific lot can be found on the datacard which accompanies each product.

Purity and Characterization::
Purity of CoA 547 was determined to be 97% by HPLC analysis. Molecular weight [M+H]+ was determined by MS to be 1506.3 (1506.4 expected).

In vitro Protein Labeling::
Reaction of CoA 547 (10 µM) with purified ACP-MBP (Maltose Binding Protein, 5 µM) and SFP Synthase (1 µM) in vitro, followed by mass spec analysis, indicated an efficiency of labeling of 44%.

Cellular Protein Labeling::
Cells transfected with pACP-GPI expressing ACP-GPI (cell surface) were labeled with 5 µM CoA 547 using SFP Synthase for 30 minutes, and visualized by confocal microscopy. Surface target was efficiently labeled.

Legal


Licenses/Patents/Disclaimers:
Notice to Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH AND DEVELOPMENT PURPOSES ONLY. Commercial use of this system or any components thereof requires a license from New England Biolabs, Inc., 240 County Road Ipswich, MA 01938. For detailed information, see www.neb.com/cia/legal/.

Protocol for CoA 547

- Instructions for Labeling on the Surface of Cells (S9349)

Overview


ACP-tag and MCP-tag fusion proteins are expressed by transient transfection. For expression of fusion proteins with the ACP-tag and MCP-tag refer to instructions supplied with the pACP and pMCP 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 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.

Protocol


   1. Dilute the Substrate Stock Solution 1:200 in medium to a final concentration of 5 µM. Mix dye 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 medium). Add MgCl2 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.
   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 30 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 from Nunc.
Wash the cells three times with tissue culture medium with serum.
Image the cells using an appropriate filter set. ACP-tag and MCP-tag fusion proteins labeled with CoA 547 should have an excitation maxi-mum at 554 nm and an emission maximum at 566 nm, and can be imaged with standard TAMRA and Cy3 filter sets.
We recommend routinely labeling one well of non-transfected or mock-transfected cells as a negative control.

Troubleshooting

No 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.

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

High Background
Background fluorescence may be controlled by reducing the concentration of substrate used, and/or 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 reduce 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.