Glutaraldehyde Conjugation Protocol

Protocol for the One-Step Glutaraldehyde Conjugation of Ferritin to (Strept)avidin [Pg.913]

This protocol is adapted from Bayer and Wilchek (1980). [Pg.913]

Prepare a solution containing 5mg/ml (strept)avidin and 25mg/ml ferritin in 0.02M sodium phosphate, 0.15 M NaCl, pH 7.4, at room temperature. Note For the coupling of other proteins to (strept)avidin, their concentration may be reduced from the 25mg/ml stated for ferritin. [Pg.913]

In a fume hood, add 10 pi of 25 percent glutaraldehyde per ml of (strept)avidin/ferritin solution. Mix well. [Pg.913]

To reduce the resultant Schiff bases and any excess aldehydes, add sodium borohydride to a final concentration of lOmg/ml. [Pg.913]

Note Some protocols do not call for a reduction step. As an alternative to reduction, add 50 xl of 0.2 M lysine in 0.5 M sodium carbonate, pH 9.5, to each milliliter of the conjugation reaction to block excess reactive sites. Block for 2 h at room temperature. Other amine-containing small molecules may be substituted for lysine—such as glycine, Tris buffer, or ethanolamine. [Pg.583]

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