Technical Notes

Site-specific covalent labeling of SNAP-tagged proteins for the measurement of binding affinities

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2 TECHNICAL NOTE ©2020 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. SNAP-Tag® labeling is an ideal tool for stable, covalent, and highly selective labeling of purified biomolecules with minimum impact on the protein's biochemical and physicochemical properties. With the intention to exploit the use of SNAP-Tag® for site-specific protein labeling in binding affinity assays, BG was conjugated with the TRIC-optimized RED 2nd Generation dye to form the RED-SNAP-Tag® 2nd Generation, included in the SNAP-Tag® Labeling Kit RED 2nd Generation (Cat# MO-LO19). To demonstrate the applicability of this labeling strategy, affinity analysis of a SNAP-tagged RNA-binding protein towards RNA molecules was performed. Results Labeling SNAP-Tag® by means of BG-derivatives is a method developed by New England Biolabs. For this study, the BG moiety was conjugated to the TRIC- optimized RED 2nd Generation fluorophore. SNAP-tagged RNA-binding protein (kindly provided by Dewpoint Therapeutics) was labeled using NanoTemper Technologies SNAP-Tag® Labeling Kit RED 2nd Generation (Cat# MO-LO19). Briefly, RED-SNAP-Tag® 2nd Generation dye was resuspended in 25 µL DMSO to yield 400 µM dye solution. For the subsequent protein labeling, 90 µL of 10 µM SNAP-tagged RNA-binding protein was mixed with 5 µL labeling buffer (20 mM Tris HCl pH 7.5, 250 mM KCl, 1 mM DTT) and 5 µL dye solution, yielding a 2:1 excess of dye over protein. The labeling reaction was carried out for 30 min at room temperature, before any excess of free dye was removed using size-exclusion chromatography and 20 mM Tris HCl pH 7.5, 250 mM KCl, 1 mM DTT, 0.05% Tween as purification buffer. For the affinity quantification of RNA-binding protein towards RNA molecules, labeled protein was added to a dilution series of either the positive or the negative control RNA at a final concentration of 20 nM using 20 mM Tris HCl pH 7.5, 250 mM KCl, 1 mM DTT, 0.05% Protein of Interest SNAP-Tag® Benzylguanine Guanine Label Labeled SNAP-tagged Protein Figure 1: Schematic representation of the SNAP-Tag® labeling approach. The reaction of SNAP-Tag® with O6-benzylguanine (BG) results in the covalent attachment of the label to the cysteine residue in the active site of the SNAP-Tag®, and the release of guanine into the solution.

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