General Questions
Q: What is the ACP-tag?
A: ACP-tag (77 amino acids) is based on Acyl Carrier Protein.
Q: How does it work?
A: ACP-tag can be labeled with Coenzyme A (CoA) derivates using ACP Synthase forming a covalent ester
bond. The small ACP-tag is particularly suited to selectively label the extracellular portion of membrane proteins.
Q: How specific is the binding of substrate to the ACP-tag?
A: This reaction is highly specific. CoA cannot label ACP-tag in the absence of SFP or ACP synthase.
Cloning and Expression
Q: What linker type and length would you recommend?
A: In general, we recommend keeping the linker as short as possible to minimize chances for non-specific protease cleavage of overly long, unstructured peptides. ACP-tag has been cloned as a fusion without a linker present.
Q: Can I clone my protein as fusion to the N- or C-terminus of the ACP-tag?
A: The ACP-tag must be cloned so that it is orientated to the extracellular surface of the plasma membrane so that the tag is accessible to its substrate and synthase.
Cell Applications
Q: Are ACP-tag substrates stable to fixation?
A: The fluorescence from ACP-tag substrates is derived from an organic fluorophore. Therefore labeled ACP-tag fusion proteins are very stable to fixations and do not lose signal intensity in contrast to some GFP spectral variants.
Q: Can ACP-tag be multiplexed with other protein labeling systems (GFP, Antibody)?
A: Yes, ACP-tag has been cloned into a construct containing GFP to compare outside and inside pools of the same protein (Jacquier et al., PNAS. 2006, George et al., JACS. 2004).
Q: Can you use ACP-tag for in vivo FRET?
A: ACP-tag has been used to label cell surfaces for FRET analysis (Meyer et al., PNAS. 2006, Meyer et al., FEBS. 2006).
Q: Does the ACP-tag labeling reaction work in Yeast?
A: Yes, if the ACP-tag is cloned so that it is orientated to the extracellular surface of the cell wall (Vivero-Pol
et al., JACS. 2005; George et al., JACS. 2004).
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