Glycan biotinylation reagents

Transfer proteins onto a nitrocellulose membrane using any appropriate procedure, including dot blotting the protein solution onto the surface. 

Incubate the membrane with the biotin derivative at a ratio of 1 ml/cc3 of membrane. 

Wash the membrane thoroughly with 0.1 M Tris, 0.15 M NaCl, pH 7.5. 

Block nonspecific sites on the membrane with an appropriate blocking component (such as BSA) and detect the biotinylated proteins using an avidin or streptavidin conjugate. 

Biotinylated oligosaccharides are convenient probes of carbohydrate interactions, because the biotin label can be captured or detected using an avidin or streptavidin derivative. For instance, immobilized streptavidin can be used to purify glycoconjugates that have been labeled 

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Figure 18.19 Biotin-PEG4-hydrazide is a hydrophilic biotinylation reagent that can be used to modify glycans or carbohydrates at their reducing end or after periodate oxidation to create aldehydes.

The biotinylated glycans on the cell surfaces subsequently may be probed with (strept)avidin reagents to detect the azido-sialic acid modifications. Alternatively, the cells may be lysed and the glycoproteins isolated using an immobilized (strept)avidin or monomeric avidin affinity resin. 

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