Labelling sugars with Fluorecent UV Labels


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We introduce to you how to label sugars which have been broken down into simple sugars. In order to chemically label sugars, reagents with fluorescence or UV absorbance capability are effective. The most general method is to introduce aromatic amine to aldehyde of the sugar chain.

Here, we introduce to you which kind of fluorescence and UV detectable ABEE (p-Amino benzoic ethyl ester or Ethyl 4-aminobenzoate) can be used to label sugars.

 

How to free sialic acid

Since sialic acid is not a reducing sugar, it is subjected to enzymatic treatment, after converting into a reducing sugar N- acyl mannosamine, before labeling.

 

1-1 Equipment and Appliances

  • Constant temperature waterbath (which can be set to 45 Ž)
  • Block heater (which can be set to 80 Ž)
  • 1.5 ml tube tabletop centrifuge
  •  

1-2 reagents used

· GlyScope sialic acid analysis kit ABEE Labeling Kit Plus S (Catalog number .JOM-J711)

· (Supplied with the kit) reaction tubes or screw-top test tube

Chloroform (0.2 per sample)

 

1-3 Procedures

1. Add 10ul of Enzyme solution (supplied with the kit) to the acid hydrolyzed samples. Close the lid and stir.

2. Centrifuge to allow liquid to drop to the bottom of the tube.

3. Incubated for one hour at 45 Ž. (Conversion of sialic acid)

4. After air-cooling, centrifuge to drop the liquid to the bottom of the tube.

5. 40ul of ABEE reagent is added, and lid is closed.

6. Stir and centrifuge to allow liquid to drop to the bottom of the tube.

7. Keep warm for 60 minutes at 80 Ž.

8. After air-cooling, add 200ul of pure water and chloroform.

9. Cap and vigorously stir and centrifuge.

10. The aqueous layer (upper layer) is collected and taken forward to HPLC analysis.

 

2 In the case of amino sugars and neutral sugars

2-1 Equipment and appliances

  • Block heater (which can be set to 80 Ž)
  • 1.5 ml tabletop centrifuge tube

 

2-2 Reagents used

  • GlyScope sialic acid analysis kit ABEE Labeling Kit Plus S (Catalog Number JOM-J711)
  • Or, GlyScope sugar labeling kit ABEE Labeling Kit (Catalog Number JOM-J710)
  • Reaction tubes or screw-top test tube (Supplied with the kit)
  • Chloroform (0.2 per sample)

 

2-3 Procedures

1. Add 10ul of pure water to acid hydrolyzed samples.

2. Add 40ul of ABEE reagent and close the lid.

3. Stir and centrifuge to allow the liquid to drop to the bottom of the tube.

4. Keep warm for 60 minutes at 80 Ž.

5. After air-cooling, add 200ul of pure water and chloroform.

6. Cap and vigorously stir and centrifuge.

7. The aqueous layer (upper layer) is collected for HPLC analysis.

 

3 When quantifying sialic acid, neutral sugars, and amino sugars at the same time

Follow steps in "2 In the case of amino sugars and neutral sugars"

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.


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