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Enzyme assay of polysialyltransferase
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Enzyme assay of polysialyltransferase

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Introduction Protocol References Credit lines
Category
Glycosyltransferases & related proteins
Protocol Name

Enzyme assay of polysialyltransferase

Authors
Kitajima, Ken *
Bioscience and Biotechnology Center, Nagoya University

Sato, Chihiro
Bioscience and Biotechnology Center, Nagoya University
*To whom correspondence should be addressed.
KeyWords
Reagents

CMP-[14C]Neu5Ac (12.4 GBq/mmol; GE Healthcare, Little Chalfont, UK)

NCAM-Fc (the soluble human neural cell adhesion molecule fused with Fc region of human IgG1)

pPROTA vector (kindly provided by Dr. John Lau, Roswell Park Cancer Institute, Buffalo, NY)

COS-1 cells (kindly provided by Dr. Karen Colley, University of Illinois at Chicago, IL)

pcDNA hPST-V5 (kindly provided by Dr. Karen Colley)

pcDNA hSTX-V5 (kindly provided by Dr. Karen Colley)

Mouse monoclonal antibody 12E3, which recognizes (Neu5Ac)n (n ≥ 5) (kindly gifted from Dr. Tatsunori Seki, Nihon Medical University School of Medicine)

anti-myc (mouse monoclonal antibody; kindly gifted from Dr. Rita Gerardy-Schahn, Medizinische Hochschule Hannover, Germany)

anti-V5 (mouse monoclonal antibody; Invitrogen/Life Technologies, Carlsbad, CA)

Instruments

Whatman 3MM paper (approximately 15 cm × 10 cm)

A tank for paper chromatography (glassware; Depth 15 cm × Width 25 cm × Height 25 cm)

BAS 2000 imaging analyzer (Fujifilm, Tokyo, Japan)

Methods
1.

Enzyme assay of polysialyltransferase

1) 

 Prepare the recombinant enzyme.

Comment 1
2) 

 Prepare the acceptor substrate.

Comment 1
3) 

 Prepare the reaction mixture (10 μL) containing 4 μL of the recombinant enzyme, 50 mM MES buffer (pH 6.0), 10 mM MnCl2, 10 mM CaCl2, 0.5% Triton CF-54, 100 mM CMP-[14C]Neu5Ac (10.7 kBq), and the acceptor substrate.

Comment 0
4) 

 Incubate at 25℃ or 37℃ for 24 h.

Comment 1
5) 

 Spot a 5-μL aliquot of reaction mixture on Whatman 3MM paper, immediately followed by spotting of 15 μL of ethanol to terminate the reactions.

Comment 0
6) 

 Develop with ethanol/1 M ammonium acetate (pH 7.5) (7:3, v/v) for 30 min.

Comment 0
7) 

 After air-drying, the amount of incorporated [14C]Neu5Ac remaining at the origin was determined by a BAS 2000 imaging analyzer.

Comment 0
8) 

 Determine the amount of enzyme used. The amount of fusion protein adsorbed to IgG-Sepharose was analyzed by Western blotting using the antibody against the epitope-tag.

Comment 0
Figure & Legends

Figure & Legends

Fig. 1A. Biosynthesis of polySia in N-linked glycan chains of the neural cell adhesion molecule (NCAM)

 

Fig. 1B. Biosynthesis of polySia in O-linked glycan chains of fish egg polysialoglycoprotein (PSGP)

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