SaHEX, an exo-β- N-acetylhexosaminidase from Streptomyces avermitilis was obtained from Nagase ChemteX Corp. The nagZ4 gene (SAV_5268) encodes SaHEX in S. avermitilis. The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number BAC72980. GlcNAc and chitin oligosaccharides, (GlcNAc) n ( n = 2–6), were obtained by the acid hydrolysis of chitin from crab shells, 18) followed by gel filtration on Cellufine Gcl-25m (JNC Co., Tokyo, Japan). Hetero-disaccharide GlcNAc-GlcN was prepared by the method of Mitsutomi et al. 19) pNP-GlcNAc was obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). TSK-GEL G2000PW and TSK Amide-80 columns used for (GlcNAc) n separation were from Tosoh Corporation (Tokyo, Japan). All other reagents were of analytical grade.
Tsk amide 80
Manufactured by Tosoh
Sourced in Japan
The TSK Amide-80 is a chromatography column designed for the separation and purification of biomolecules. It features an amide-based stationary phase that provides a unique selectivity for the analysis of proteins, peptides, and other biological compounds.
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3 protocols using tsk amide 80
1
Characterization of SaHEX Enzyme from Streptomyces avermitilis
2
HILIC Fractionation and LC-MS/MS Analysis of Phosphorylated Peptides
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The fractions containing mono-phosphorylated, deglycosylated and unmodified peptides were fractionated prior to nano LC-MS/MS analysis using HILIC as described previously [63 (link), 64 (link)]. Peptides were dissolved in 90% ACN, 0.1% TFA (solvent B) and loaded onto a 450 μm OD × 320 μm ID × 17 cm micro-capillary column packed with TSK Amide-80 (3 μm; Tosoh Bioscience) using an Agilent 1200 Series HPLC (Agilent). The peptides were separated using a gradient from 100–60% solvent B (A = 0.1% TFA) in 30 min at a flow-rate of 6 μl/min. Fractions were collected every 1 min based on the UV chromatogram. Subsequently, the peptide fractions were dried by vacuum centrifugation.
3
HILIC Fractionation for Phosphopeptide Analysis
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The unmodified and phosphorylated peptide samples were fractionated prior to nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis using HILIC as described previously [60 (link), 61 (link)]. Peptides were dissolved in 90% ACN, 0.1% TFA (solvent B) and loaded onto a 450 μm OD × 320 μm ID × 17 cm micro-capillary column packed with TSK Amide-80 (3 μm; Tosoh Bioscience) using an Agilent 1200 Series HPLC (Agilent). The peptides were separated using a gradient from 100–60% solvent B (A = 0.1% TFA) in 30 min at a flow-rate of 6 μl/min. Fractions were collected every 1 min based on the UV chromatogram. Subsequently, the peptide fractions were dried by vacuum centrifugation.
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