Coupling through amino groups

1. Glutardldehyde coupling is not suggested for peptides with lysine or N-terminus peptides.

2. Dissolve 5 mg of Keyhole limpet hemocyanin (KLH) in 5 ml PBS.

3. Dissolve 5 mg peptides to 5 ml of KLH/PBS solution.

4. Adjust pH to 9.0 with 0.1 M LiOH.

5. Add dropwise 6.25% glutardldehyde to achieve a final concentration of 1%.

6. Incubate at 4 °C for 1 hour with gentle agitation.

7. Remove unbound peptide by size exclusion column (e.g. Sephadex G25, PD-10) or dialysis.

8. For a two-step protocl, add glutardldehyde to peptides. Remove glutardldehyde using a Sephadex G10 column and them mix activated peptides to KLH.

Coupling through cysteines

1. The peptide must have either a N-termiual or C-terminal cysteine.

2. Prepare fresh MBS: Dissolve 3 mg m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) in 200 ml Dimethyl Formamide (DMF).

3. Add 70 ml MBS/DMF to KLH solution (5 mg keyhole limpet hemocyanin (KLH) in 0.5 ml 10 mM Phosphate Buffer, pH 7.0).

4. Stir gently at room temperature for 30 minutes.

5. Remove free corsslinker by a size exclusion column (e.g. Sephadex G25, PD-10).

Equilibrate the column with 50 mM Phosphate Buffer (pH 6.0). Load KLH reaction mixture. Elute with 50 mM Phosphate Buffer (pH 6.0).

6. Dissolve 5 mg synthetic peptide in an appropriate buffer(e.g., PBS or 6M guanidine-HCl/10 mM phosphate buffer, pH7.0).

7. Add peptide solution to MB/KLH conjugate.

8. Adjust pH to 7.3 with 0.1M HCl or 0.1M NaOH.

9. Stir for 3 hours at room temperature.

10. Remove unbound peptide by another size exclusion column (e.g. Sephadex G25, PD-10).

Equilibrate the column with ddH₂O. Load KLH reaction mixture. Elute with ddH₂O.

11. Concentrate the conjugate.