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The activity of the SNAP tag was confirmed by labeling the Cdc10-SNAP with BG-TMR (New England Biolabs).
For this, we exploited the SNAP tag, which consists of a 20 kD suicide enzyme that has been mutated to use benzylguanine as a substrate [29].
Here, the SNAP tag replaced the S tag of the Cdc10 subunit.
We subsequently applied our established purification protocol to SNAP tag modified septin rods.
We provide methods for purifying SNAP tag labeled septin rods in high yield and purity.
Individual cargo at the surface could be followed using a reducible fluorescent ligand for the SNAP tag.
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The SNAP-tag is fused to the GFP-tagged Golgi membrane anchor from galactosyl transferase for proper targeting to trans-Golgi/TGN membranes.
We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells.
Recently, various fluorogenic probes for SNAP-tag based protein labeling were reported.
SNAP-tag (20 kDa) was derived from the human DNA repair protein O 6-alkylguanine-DNA alkyl-transferase (AGT).
This method also has several advantages over currently employed chemical-based labeling techniques, such as SNAP-tag and CLIP-tag.
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