Periodate Oxidation of the Antibody

1. Prepare the antibody for periodate oxidation. Dialyze the glycosylated antibody at 2-10 mg/mL overnight at 4°C vs 100 ml acetate buffer (pH 6.0) and keep the solution on ice during the oxidation step. Because this reaction is light sensitive, amber or opaque containers should be used for all reagents.

2. Oxidize the antibody. Prepare a 20-mM solution of sodium metaperiodate in ice-cold acetate buffer (pH 6.0) and add an equal volume drop-wise to the antibody while stirring. Incubate the mixture for 120 min on ice.

3. Desalt the oxidized antibody. To remove iodate and formaldehyde byproducts, dialyze the oxidized antibody overnight at 4°C in the dark versus an amine-free buffer at pH 6.0-7.2 or use gel filtration. For optimum biotinylation, the oxidized antibody should be concentrated to approx 10 mg protein/mL (see Note 5).

0 0

Post a comment