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Sugar chain detection by the phenol-sulfuric acid method

This method is very simple and easy. No pretreatment is necessary. The amount of neutral sugars in glycoproteins can be measured by colometrically (Hodge, JE and Hofreiter, BT, Method in Carbohydrate Chemistry, 1, 338 (1962))

 

1. Measurement Range

Neutral sugars 0-100μg / mL

 

2. Equipment and Appliances

l  5% Phenol reagent (W / V) aqueous phenol solution

l  Concentrated sulfuric acid

l  Standard monosaccharide (i.e mannose)

l  105 × 15mm Test tubes

l  Spectrophotometer (which can measure at 490nm)

 

3. Operation

1. Add 500μL of sample aqueous solution or standard monosaccharide aqueous solution in a test tube

2. add 500μL of phenol reagent, and stir well

3. Add 2.5mL  of concentrated sulfuric acid, and immediately stirr for 10 seconds vigorously (please note that heat will be generated)

4. Leave at room temperature for more than 20 minutes

5. Measure absorption at 490nm with a spectrophotometer

 

4. Making Decisions

l  If  absorption is measured at 490nm, the protein sample contains neutral sugars which can be assumed to be glycoproteins.

l  By comparing with the calibration curve of standard monosaccharides, it is possible to calculate the approximate sugar content of glycoprotein in the sample(neutral sugars).

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