Two-Step Glutaraldehyde Protocol

Dissolve the enzyme at a concentration of lOmg/ml in 0.1 M sodium phosphate, 0.15M NaCl, pH 6.8. 

Purify the activated enzyme from excess glutaraldehyde by gel filtration using a desalting resin or by dialysis against PBS, pH 6.8. 

Dissolve the antibody to be conjugated at a concentration of lOmg/ml in 0.5M sodium carbonate, pH 9.5. Mix the activated enzyme with the antibody at the desired molar ratio to effect the conjugation. Mixing the equivalent of 4 mg of enzyme per mg of antibody usually results in acceptable conjugates. 

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

Note Some protocols avoid a reduction step. As an alternative to reduction, add 50 pi of 0.2M lysine in 0.5M 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. 

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Protocol for the Two-Step Glutaraldehyde Conjugation of Enzymes to (Strept)avidin... 

The following protocol describes the two-step method wherein the liposome is glutaraldehyde-activated, purified away from excess cross-linker, and then coupled to a protein by reductive animation (Fig. 352). 

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