Enzyme assay of O-fucose, fringe, and O-GlcNAc transferases modifying epidermal growth factor domains ~Fringe assay

Authors：

Category

Glycosyltransferases & related proteins

Protocol Name

Authors

Okajima, Tetsuya *
Department of Biochemistry II, Nagoya University Graduate School of Medicine

Furukawa, Koichi
Department of Biochemistry II, Nagoya University Graduate School of Medicine

*To whom correspondence should be addressed.

KeyWords

Epidermal Growth Factor Notch O-fucose

Reagents

●	Notch EGF repeats with FLAG tag (N-EGF:FLAG) isolated from S2 cells
●	anti-FLAG antibody-conjugated agarose (Sigma-Aldrich, St. Louis, MO)
●	1x Fringe assay buffer (50 mM Hepes, pH 7.0, 150 mM NaCl, 50 mM MnCl₂)
●	Purified Fringe:6xHis (Moloney et al., 2000)
●	HBSS (Invitrogen/Life Technologies, Carlsbad, CA)

Methods

Enzyme assay of fringe

1)	N-EGF:FLAG is imobilzed on anti-FLAG antibody-conjugated agarose.	Comment 0

2)	The beads are washed once with 1x Fringe assay buffer.	Comment 0

3)	For the assay, 18 μM UDP-[¹⁴C]GlcNAc (GE Healthcare, Little Chalfont, UK), the FLAG beads, and the enzyme were mixed in 1x Fringe assay buffer in a volume of 50 μL.	Comment 0

4)	After 4 h of incubation at 30˚C with constant gentle mixing, the beads are washed four times with HBSS.	Comment 0

Radioactivity on the beads is measured using a liquid scintillation counter.

Comment 0

Figure & Legends

Fig. 1. Assay for Fringe activity

In vitro glycosylation of N-EGF:FLAG by Fringe. N-EGF:FLAG was produced from wild type of OFUT1-depleted S2 cells. The amount of product formed is indicated by the amount of GlcNAc transferred to equivalent amounts of N-EGF:FLAG (Okajima et al., 2003).

This figure was originally published in "Experimental Glycoscience -Glycobiology" edited by Taniguchi N. et al. Springer Japan KK. 2008, pp.33-35 (Okajima T. Part1: Section I).

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Date of registration：2013-12-25 09:59:18