Succinylation-Alcian Blue Staining for Mucins

Authors：

Category

Isolation & structural analysis of glycans

Protocol Name

Authors

Dong, Weijie
Advanced Glycoscience Research Group, Bioproduction Research Institute, AIST

Kameyama, Akihiko *
Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology (AIST)

*To whom correspondence should be addressed.

KeyWords

mucin PVDF membrane staining

Reagents

●	Succinic anhydride, (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan)
●	Acetone, pyridine, Acetonitrile, and cis-4-nonene, (Wako Pure Chemical Industries, Ltd., Osaka, Japan)
●	Alcian blue 8GX, (Sigma-Aldrich, St. Louis, MO)

Instruments

●	Heat block
●	Glass dish
●	Image analyzer (LAS-1000 plus, Fujifilm, Tokyo, Japan)

Methods

This procedure describes for staining mucins blotted onto the PVDF membrane or separated by PVDF membrane electrophoresis²⁾. (Fig.1)

1)	Immerse the membranes in acetone for 30 min.	Comment 1

2)	Dry the membranes by heating at 80˚C for 20 min using a heating block.	Comment 0

3)	Prepare the reaction solution for the succinylation as follows: dissolve 500 mg of succinic anhydride in 5 mL of acetonitrile containing pyridine in a 4:1 ratio (v:v).	Comment 1

4)	Immerse the dried membranes in the freshly prepared reaction solution in a sealed glass dish and incubate for 2 h at room temperature.	Comment 0

5)	Transfer the membranes into 0.1% (w/v) Alcian blue 8GX dissolved in 0.1% acetic acid, and gently shake for 20 min.	Comment 0

6)	Pull out the membrane from Alcian blue solution, and wash with methanol for a few minutes to remove the background color.	Comment 0

For acquiring quantitative data, make the stained membrane transparent by immersion in cis-4-nonene, and perform densitometry with an image analyzer.

Comment 1

Notes

Safety concerns

This procedure should be performed in a fume hood.

Figure & Legends

Fig. 1. Principle of succinylation-Alcian blue staining

Carboxyl groups are introduced to glycan cluster of neutral mucins by succinylation. The succinylated mucins can be by stained Alcian blue. Succinyl groups are easily removed from glycans during alkaline ß-elimination which is a glycan releasing reaction for O-linked glycans.

Reprinted with permission from Anal. Chem. 84(20), 8461–8466, Dong, W., Matsuno, Y-K., Kameyama, A. A procedure for Alcian blue staining of mucins on polyvinylidene difluoride membranes. Copyright 2012 American Chemical Society.

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Date of registration：2015-03-04 13:39:00