Biotinylation glycans

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. 

In some cases, the ability to modify glycans at the reducing end without reduction preserves the carbohydrate s native structure sufficiently to allow interactions with proteins that would otherwise not interact if the bond were reduced. Therefore, depending on the ultimate use of the biotinylated carbohydrate, using a hydrazide mediated conjugation process can have advantages over the use of amine-biotin compounds. 

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 following protocol describes a method for the periodate oxidation of a glycoprotein followed by biotinylation of the resultant aldehydes using hydrazide-PEG4-biotin. Chapter 1, Section 4.6 describes an alternative protocol for the modification of glycans at their reducing ends with hydrazide compounds. 

Leteux, C., Childs, R.A., Chai, W., Stoll, M.S., Kogelberg, H., and Feizi, T. (1998) Biotinyl-L-3-(2-naph-thyl)-alanine hydrazide derivatives of N-glycans Versatile solid-phase probes for carbohydrate-recognition studies. Glycobiology 8, 227-236. 

Prepare glycan-binding protein solutions at desired concentrations in TSMBB. The GBPs are pre-labeled with either biotin or fluorescent dyes. We generally use biotinylated lectins at 10 pg/mL or lower (see Note 7). 

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