- This topic has 4 replies, 3 voices, and was last updated 8 months, 2 weeks ago by Martin Möckel.
March 19, 2020 at 11:42 #2863
although we are almost shut down, we still wprk on emergency projects.
In this context we are asked to produce a protein with a tag that can be fluorecently labelled for cell sorting. So far we have provided Sortase tag, but I heard from our users, that efficiency is protein dependent.
Do you have any other or better ideas?
Thank you very much in advance
March 19, 2020 at 12:17 #2864
Hidde Ploegh’s lab published this one recently.
Moreover, what about Halotags ?
March 19, 2020 at 12:38 #2867
I think Snap and Halo tags are good for stoichiometric labelling with bright dyes and they are reasonably small, too. For the Snap tag, NEB sells all the dyes that can be conjugated to it.
Is bioorthogonal labelling an option (e.g. using NHS or maleimide-activated dyes)? Sometimes we are lucky here and work with proteins that do not have cysteines, so we can add one at the N/or C-terminus for labelling.
There might also be the (more complicated) route of using an N-or C-terminal AVI tag in combination with BirA-ligase expression for in vivo biotinylation of the protein and then using fluorescently labelled streptavidin in a second incubation step with the cells that are prepared for FACS.
- This reply was modified 8 months, 2 weeks ago by Martin Möckel.
March 19, 2020 at 12:42 #2869
Hi Svend and Martin
thanks for the advice.
I haven’t thought about Cys, thats another alternative.
If we are using Snap, any thing important to know when designing the construct?
March 19, 2020 at 15:48 #2875
There should be no special considerations for tag order. If you want to make sure only full length protein gets labelled, you could put the SNAP tag on the C-terminus. We had His-POI-SNAP once, but all configurations are fine. Reaction conditions are on the NEB page, but the benzylguanin reacts in most protein-friendly environments.
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