MBS Coupling Method see Notes

1. Dissolve 15-20 mg of carrier protein in a small amount of phosphate-buffered saline (about 1 mL).

2. Dissolve 5 mg of MBS in a small amount of dimethylformamide (about 0.75 mL) or for Sulfo-MBS, dissolve in a small amount of sterile water.

3. When crosslinker and carrier are completely dissolved, mix well and leave at room temperature for 1 h.

4. Desalt on a 20-mL Sephadex G25 column using 0.1 M sodium phosphate buffer, pH 6.0. Collect 2-mL fractions. Read the optical density (OD) of the fractions at 280 nm. Keep the two fractions with the highest OD280.

5. Meanwhile, reduce the peptide.

a. Make up fresh 5 mg/mL solution of sodium borohydride and store on ice.

b. Dissolve 15-20 mg of peptide in minimum amount of 0.1 M borate buffer, pH 8.0.

c. Add 100 mL of sodium borohydride to the dissolved peptide, mix well, and stand on ice for 5 min.

d. Lower pH by adding 1 M HCl (approx five drops), mix, and leave on ice for a further 5 min.

e. Add equal number of drops of 1 M NaOH and check that the pH is between 6 and 7. If not, then adjust with 1 M NaOH or 1 M HCl. (10 mL is approx 0.5 of a pH unit).

6. Add desalted crosslinker/carrier to reduced peptide and leave overnight at room temperature.

7. If the conjugate becomes insoluble, precipitate completely with 4-5 vol of ice-cold acetone at -70°C for 30 min. Briefly warm at room temperature. Pour off the supernatant and air dry. Resuspend in saline as in Subheading 3.1.4. Alternatively, if the conjugate remains soluble, then desalt the solution on a 20-mL Sephadex G25 column using ammonium hydrogen carbonate buffer, pH 7.5. Collect 2-mL fractions and pool those of OD280 > 0.4. Conjugates can be stored at -20°C and rehomogenized before use.

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