Hi Guillermo,

We have been using post-translational labelling systems for a while now, although mostly for single molecule tracking experiments, rather than for super-resolution.For some reason the SMT community have converged on HaloTags, but I think that the same arguments apply to SnapTags,  We did not encounter major problems using these systems - and we recently managed to get our protein of interest endogenously labelled with HaloTag. A minor drawback is that you might have to adjust the labelling protoccol for your settings. A second one is that by performing a "pulse" labelling you will not be allowed to follow your protein of interest for a long time after labelling (especially if the protein turnover is fast).

The advantages as you say is the possibility to select the dye for the labelling, which being organic dyes are typically brighter and photostable than FPs. Plus, there's intense active research on developing ligands for Halos- and Snaps-: in recent years the labs of Kai Johnson and Luke Lavis have developed some very interesting fluorescent ligands ranging from fluorogenic probes for STED to photoactivatable/photoswitchable dyes for PALM/STORM.

My feeling is that the answer depends on what kind of super-resolution microscopy you would like to use. If it's single molecule localization based (PALM/STORM/etc), then the two possibilities are either tagging with a photoactivatable/photoswitchable fluorescent protein - but then you cannot do much more than PALM/STORM with your cells, or using SnapTags with a photoswitchable dye. If the technique is SIM  then pretty much any solution might work. If the technique is STED, then I guess you would have to select the probe depending on the depletion laser that you have on your set-up, organic dyes should work better in this case - but it is just a guess, because i do not have a direct experience with STED.

Hope this helps,

Davide

Hi Guillermo,

We have been using post-translational labelling systems for a while now, although mostly for single molecule tracking experiments, rather than for super-resolution.For some reason the SMT community have converged on HaloTags, but I think that the same arguments apply to SnapTags,  We did not encounter major problems using these systems - and we recently managed to get our protein of interest endogenously labelled with HaloTag. A minor drawback is that you might have to adjust the labelling protoccol for your settings. A second one is that by performing a "pulse" labelling you will not be allowed to follow your protein of interest for a long time after labelling (especially if the protein turnover is fast).

The advantages as you say is the possibility to select the dye for the labelling, which being organic dyes are typically brighter and photostable than FPs. Plus, there's intense active research on developing ligands for Halos- and Snaps-: in recent years the labs of Kai Johnson and Luke Lavis have developed some very interesting fluorescent ligands ranging from fluorogenic probes for STED to photoactivatable/photoswitchable dyes for PALM/STORM.

My feeling is that the answer depends on what kind of super-resolution microscopy you would like to use. If it's single molecule localization based (PALM/STORM/etc), then the two possibilities are either tagging with a photoactivatable/photoswitchable fluorescent protein - but then you cannot do much more than PALM/STORM with your cells, or using SnapTags with a photoswitchable dye. If the technique is SIM  then pretty much any solution might work. If the technique is STED, then I guess you would have to select the probe depending on the depletion laser that you have on your set-up, organic dyes should work better in this case - but it is just a guess, because i do not have a direct experience with STED.

Hope this helps,

Davide

Hi Davide,

Thanks a lot for your reply, it is exactly what I needed, I think I will likely settle for the SNAP-Tag as I will be doing STED imaging.

Thanks again for your helpful answer,

best regards, 

Guillermo