One-Step Glutaraldehyde Protocol

Prepare a solution containing 2mg/ml antibody and 5mg/ml enzyme in 0.02M sodium phosphate, 0.15 M NaCl, pH 7.4, chilled to 4°C. 

In a fume hood, add 10 pi of 25 percent glutaraldehyde (Sigma) per ml of antibody/enzyme solution. Mix well. 

To reduce the resultant Schiff bases and any excess aldehydes, add sodium borohydride (Aldrich) to a final concentration of lOmg/ml. 

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

To remove any insoluble polymers that may have formed, centrifuge the conjugate or filter it through a 0.45 pm filter. Purify the conjugate by gel filtration or dialysis using PBS, pH 7.4. 

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