General Carbohydrate Detection on Western Blots

Carbohydrates are easily oxidized by periodate to aldehydes which react with primary amines or hydrazines. If the formed hydrazide carries a specific ligand, e.g., biotin or digoxigenin, these ligands immobilized via the blotted macromolecule are very sensitively detected by the respective enzyme conjugates. 

Agitate the unblocked membrane after electrotransfer in Soln. A three times for 10 min. Equilibrate the membrane in Soln. B for 1 min, discard Soln. B and incubate with Soln. C (about 0.5 ml/cm ) for 20 min. Wash three times with Soln. A. 

Add a sufficient amount of the hydrazide (e.g., 0.2 pg/ml of digoxigenin hydrazide (digoxigenin-succinyl-e-aminocaproic acid hydrazide) or 25pg/ml of Biotin-LC-Hydrazide ) and incubate for 1 h at RT. Wash three times for 10 min with Soln. D and block the membranes for 15 min with an appropriate blocking solution, e.g., 1% non-fat dry milk in Soln. D. Wash with Soln. D. 

Incubate with the corresponding enzyme conjugate (e.g., anti-digoxigenin AP conjugate or streptavidin-HRP) for 30 min, wash carefully and perform the staining as described in Protocols 2.5.5 and 2.5.4. 

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