Specificity profiling reactions were initiated by addition of subtiligase or variant (1 µM) to a reaction mixture containing peptide library (1 mM), biotinylated peptide ester 1 (0.2 mM), and 100 mM tricine, pH 8.0. After 1 h, reactions were quenched by addition of 1 volume of 8 M guanidine hydrochloride. Biotinylated peptides were enriched on High-Capacity Neutravidin resin (Thermo Fisher Scientific) (0.25 mL of 50% resin slurry). The resin was washed five times with 0.5 mL 4 M guanidine hydrochloride and five times with TEV elution buffer (100 mM ammonium bicarbonate, 2 mM DTT). The resin was resuspended in 0.25 mL TEV elution buffer and incubated with TEV protease (10 µg) for 2 h to selectively elute biotinylated peptides. Resin was removed from the eluted peptides using a spin filter. The solution containing the eluted peptides was adjusted to 5% TFA, incubated at room temperature for 10 min, and spun at 20,000 × g to remove precipitated TEV protease. Peptides were then desalted on C18 OMIX tips, dried, dissolved in 10 µL 0.1% formic acid, and analyzed by LC-MS/MS.
High capacity neutravidin resin
Manufactured by Thermo Fisher Scientific
The High-Capacity Neutravidin resin is a solid-phase affinity chromatography matrix used for the purification and immobilization of biotinylated molecules. It offers a high binding capacity for biotin-labeled proteins, nucleic acids, and other biomolecules. The resin is designed to provide efficient and selective capture of biotinylated targets.
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Lab products found in correlation
2 protocols using high capacity neutravidin resin
1
Specificity Profiling of Subtiligase Variants
2
Specificity Profiling of Subtiligase Variants
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Specificity profiling reactions were initiated by addition of subtiligase or variant (1 µM) to a reaction mixture containing peptide library (1 mM), biotinylated peptide ester 1 (0.2 mM), and 100 mM tricine, pH 8.0. After 1 h, reactions were quenched by addition of 1 volume of 8 M guanidine hydrochloride. Biotinylated peptides were enriched on High-Capacity Neutravidin resin (Thermo Fisher Scientific) (0.25 mL of 50% resin slurry). The resin was washed five times with 0.5 mL 4 M guanidine hydrochloride and five times with TEV elution buffer (100 mM ammonium bicarbonate, 2 mM DTT). The resin was resuspended in 0.25 mL TEV elution buffer and incubated with TEV protease (10 µg) for 2 h to selectively elute biotinylated peptides. Resin was removed from the eluted peptides using a spin filter. The solution containing the eluted peptides was adjusted to 5% TFA, incubated at room temperature for 10 min, and spun at 20,000 × g to remove precipitated TEV protease. Peptides were then desalted on C18 OMIX tips, dried, dissolved in 10 µL 0.1% formic acid, and analyzed by LC-MS/MS.
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