Conjugation of Peptides

1. KLH: purchased as a solution in 50% glycerol (Calbiochem) and stored at 4°C. If obtained as an ammonium sulfate suspension the KLH will require extensive dialysis against borate buffered saline (20 ml Na Borate/144 ml NaCl) containing 50% glycerol.

2. Glutaraldehyde (Sigma, Grade 1) stock: is a 25% solution divided into 1-mL aliquots. It is stored at -20°C and never re-frozen.

3. Sodium bicarbonate stock (10X): a 1-M solution adjusted to pH 9.6 with HCl.

4. Glycine ethyl ester hydrochloride (Sigma): make up as a 1-M stock and adjust to pH 8.0 with NaOH.

5. MBS "Sulfo-MBS" version of this reagent is water-soluble and, therefore, preferable (Pierce).

6. 0.1 M Sodium phosphate buffer, pH 6.0: mix 12 mL of 1 M disodium hydrogen phosphate with 88 mL of 1 M sodium dihydrogen phosphate and make up to 1 L with water.

7. Sephadex G25 (APBiotech).

8. Sodium borohydride.

9. Borate buffer: 0.1-M boric acid solution adjusted to pH 8.0 with NaOH.

12. Acetone.

14. Ammonium hydrogen carbonate, pH 7.5.

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