Hydrazide-PEG4-biotin

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. 

Dissolve a glycoprotein to be oxidized in 0.1 M sodium acetate, pH 5.5 (oxidation buffer), at a concentration of 2-10mg/ml. PBS at physiological pH may be used for this reaction, as well. The use of cold buffers for the oxidation step will limit the extent of carbohydrate oxidation and the potential for protein oxidation. 

Dissolve sodium meta-periodate in oxidation buffer at a concentration of 20 mM. Protect from light. 

Add an equal volume of the glycoprotein solution to the periodate solution with mixing. 

React for 10-20 minutes with gentle mixing and protected from light. 

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BupH MES buffered saline packs (Thermo Scientific, cat. no. 28390) EZ-Link hydrazide—PEG4—biotin (Thermo Scientific, cat. no. 21360) Dimethyl sulfoxide (DMSO Sigma, cat. no. D8418)... 

Resuspend the hydrazide—PEG4—biotin at 250 mM in DMSO add 396 pL of DMSO to the vial of hydrazide. Dilute this stock solution 1 5 in DMSO to obtain 50 mM hydrazide—PEG4—biotin. Store the 250 mM stock solution at —20 °C. 

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.

Figure 18.20 Biotin-PEG4-hydrazide reacts with aldehyde-containing molecules to form a hydrazone linkage.

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