1. Weigh out the peptide and an equal weight of KLH (or thyroglobu-lin) carrier. This gives an approximate ratio of 40-150 molecules of peptide to each molecule of carrier (2 mg of peptide per animal is ample).
2. Dissolve the peptide and carrier protein in 0.1 M (1X) sodium bicarbonate using 1 mL for every 2 mg of carrier protein.
3. Thaw out a fresh vial of glutaraldehyde and add to the peptide-car-rier solution to a final concentration of 0.05%. Mix in a glass tube, stirring with a magnetic stirring bar; keep at room temperature overnight in the dark (wrap the tube in foil). The solution will usually turn a pale yellow color. Occasionally the solution will turn pale brown or orange—this reflects the fact that peptide preparations sometimes contain traces of chemical scavenger reagents used in the final cleavage of the peptide from the resin and is not a cause for concern.
4. Either: dialyze against double distilled water for 12 h and lyophilize the coupled carrier. Assess yield by weighing the lyophilized material to determine the percentage of peptide coupled.
Or, because coupling efficiency is usually reasonable, and not too critical, it is easier to do the following: add 1 M glycine ethyl ester to a final concentration of 0.1 M and leave for 30 min at room temperature. Then, precipitate the coupled carrier with 4-5 vol of ice-cold acetone at -70°C for 30 min. Briefly warm at room temperature and pellet the protein at 10,000g for 10 min at room temperature, pour off the acetone, air dry the pellet, and redisperse it in saline at 1 mg carrier/mL. As the pelleted protein is rather sticky, this is best done using a Dounce™ homogenizer. Conjugates can be stored at -20°C and rehomogenized before use.
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