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Hydrazinolysis using hydrazine monohydrate
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Hydrazinolysis using hydrazine monohydrate

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Introduction Protocol References Credit lines
Category
N-Glycans
Protocol Name

Hydrazinolysis using hydrazine monohydrate

Authors
 Nakakita, Shin-ichi *
Department of Functional Glycomics, Life Science Research Center, Kagawa University

Sumiyoshi, Wataru
Department of Glyco-Bioindustry, Life Science Research Center, Kagawa University

Natsuka, Shunji
Department of Biology, Faculty of Science, Niigata University
*To whom correspondence should be addressed.
KeyWords
Reagents
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Hydrazine Monohydrous (Wako Pure Chemical Industries, Ltd. Osaka, Japan)

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Sodium Hydrogen Carbonate (Wako Pure Chemical Industries, Ltd.)

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Acetic anhydride (Wako Pure Chemical Industries, Ltd.)

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Dowex 50 W (The Dow Chemical Company, Midland, MI)

Instruments
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Speed Vac Concentrator (Thermo Fisher Scientific Inc., , Waltham, MA)
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Block incubator (Astec Co., Ltd., Fukuoka, Japan)
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Rotary oil pump connected to cold trap (Yamato Scientific Co., Ltd. Tokyo, Japan)
Methods
1.

Hydrazinolysis using hydrazine monohydrate

1) 

 Put lyophilized sample of glycoprotein or tissue (2 mg in this protocol’s scale) at the bottom of a screw cap tube.

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2) 

 Add 200 μL of hydrazine monohydrous in the tube.

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3) 

 Mix hydrazine and powder.

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4) 

 Heat at 80°C, 10 h.

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5) 

 Remove hydrazine in vacuo using a rotary pump connected with cold glass trap.

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6) 

 Add 0.2 mL saturated sodium bicarbonate solution and 0.016 mL acetic anhydride at 0°C for 5 min.

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7) 

 Add 0.2 mL saturated sodium bicarbonate solution and 0.016 mL acetic anhydride at 0°C for 30 min.

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8) 

 Add 1 g of Dowex 50 W-X2 (H+ form).

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9) 

 Pour the mixture into a small glass column.

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10) 

 Wash with 5 column volumes of water.

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11) 

 Combine the eluate and the washings, and then concentrate, and lyophilize.

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Initial amount

1 mg – 1 g

Produced amount

1 ng – 1 mg

Discussion

This method clearly indicates that the large-scale preparation of N-glycan from glycoproteins is readily achievable using the relatively safe chemical, hydrazine monohydrate.

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Date of registration：2013-11-08 10:48:10
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